Journal articles on the topic 'Protein Kinase, c-Src'
To see the other types of publications on this topic, follow the link: Protein Kinase, c-Src.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'Protein Kinase, c-Src.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
Sabe, H., M. Okada, H. Nakagawa, and H. Hanafusa. "Activation of c-Src in cells bearing v-Crk and its suppression by Csk." Molecular and Cellular Biology 12, no. 10 (October 1992): 4706–13. http://dx.doi.org/10.1128/mcb.12.10.4706-4713.1992.
Full textAbstract:
The protein product of the CT10 virus, p47gag-crk (v-Crk), which contains Src homology region 2 (SH2) and 3 (SH3) domains but lacks a kinase domain, is believed to cause an increase in cellular protein tyrosine phosphorylation. A candidate tyrosine kinase, Csk (C-terminal Src kinase), has been implicated in c-Src Tyr-527 phosphorylation, which negatively regulates the protein tyrosine kinase of pp60c-src (c-Src). To investigate how c-Src kinase activity is regulated in vivo, we first looked at whether v-Crk can activate c-Src kinase. We found that cooverexpression of v-Crk and c-Src caused elevation of c-Src kinase activity, resulting in an increase of tyrosine phosphorylation of cellular proteins and morphological transformation of rat 3Y1 fibroblasts. v-Crk and c-Src complexes were not detected, although v-Crk bound to a variety of tyrosine-phosphorylated proteins in cells overexpressing v-Crk and c-Src. Overexpression of Csk in these transformed cells caused reversion to normal phenotypes and also reduced the level of c-Src kinase activity. However, Csk did not cause reversion of cells transformed by v-Src or c-Src527F, in which Tyr-527 was changed to Phe. These results strongly suggest that Csk acts on Tyr-527 of c-Src and suppresses c-Src kinase activity in vivo. Because Csk can suppress transformation by cooverexpression of v-Crk and c-Src, we suggest that v-Crk causes activation of c-Src in vivo by altering the phosphorylation state of Tyr-527.
2
Sabe, H., M. Okada, H. Nakagawa, and H. Hanafusa. "Activation of c-Src in cells bearing v-Crk and its suppression by Csk." Molecular and Cellular Biology 12, no. 10 (October 1992): 4706–13. http://dx.doi.org/10.1128/mcb.12.10.4706.
Full textAbstract:
The protein product of the CT10 virus, p47gag-crk (v-Crk), which contains Src homology region 2 (SH2) and 3 (SH3) domains but lacks a kinase domain, is believed to cause an increase in cellular protein tyrosine phosphorylation. A candidate tyrosine kinase, Csk (C-terminal Src kinase), has been implicated in c-Src Tyr-527 phosphorylation, which negatively regulates the protein tyrosine kinase of pp60c-src (c-Src). To investigate how c-Src kinase activity is regulated in vivo, we first looked at whether v-Crk can activate c-Src kinase. We found that cooverexpression of v-Crk and c-Src caused elevation of c-Src kinase activity, resulting in an increase of tyrosine phosphorylation of cellular proteins and morphological transformation of rat 3Y1 fibroblasts. v-Crk and c-Src complexes were not detected, although v-Crk bound to a variety of tyrosine-phosphorylated proteins in cells overexpressing v-Crk and c-Src. Overexpression of Csk in these transformed cells caused reversion to normal phenotypes and also reduced the level of c-Src kinase activity. However, Csk did not cause reversion of cells transformed by v-Src or c-Src527F, in which Tyr-527 was changed to Phe. These results strongly suggest that Csk acts on Tyr-527 of c-Src and suppresses c-Src kinase activity in vivo. Because Csk can suppress transformation by cooverexpression of v-Crk and c-Src, we suggest that v-Crk causes activation of c-Src in vivo by altering the phosphorylation state of Tyr-527.
3
Brott, B. K., S. Decker, M. C. O'Brien, and R. Jove. "Molecular features of the viral and cellular Src kinases involved in interactions with the GTPase-activating protein." Molecular and Cellular Biology 11, no. 10 (October 1991): 5059–67. http://dx.doi.org/10.1128/mcb.11.10.5059-5067.1991.
Full textAbstract:
GTPase-activating protein (GAP) enhances the rate of GTP hydrolysis by cellular Ras proteins and is implicated in mitogenic signal transduction. GAP is phosphorylated on tyrosine in cells transformed by Rous sarcoma virus and serves as an in vitro substrate of the viral Src (v-Src) kinase. Our previous studies showed that GAP complexes stably with normal cellular Src (c-Src), although its association with v-Src is less stable. To further investigate the molecular basis for interactions between GAP and the Src kinases, we examined GAP association with and phosphorylation by a series of c-Src and v-Src mutants. Analysis of GAP association with c-Src/v-Src chimeric proteins demonstrates that GAP associates stably with Src proteins possessing low kinase activity and poorly with activated Src kinases, especially those that lack the carboxy-terminal segment of c-Src containing the regulatory amino acid Tyr-527. Phosphorylated Tyr-527 is a major determinant of c-Src association with GAP, as demonstrated by c-Src point mutants in which Tyr-527 is changed to Phe. While the isolated amino-terminal half of the c-Src protein is insufficient for stable GAP association, analysis of point substitutions of highly conserved amino acid residues in the c-Src SH2 region indicate that this region also influences Src-GAP complex formation. Therefore, our results suggest that both Tyr-527 phosphorylation and the SH2 region contribute to stable association of c-Src with GAP. Analysis of in vivo phosphorylation of GAP by v-Src mutants containing deletions encompassing the SH2, SH3, and unique regions suggests that the kinase domain of v-Src contains sufficient substrate specificity for GAP phosphorylation. Even though tyrosine phosphorylation of GAP correlates to certain extent with the transforming ability of various c-Src and v-Src mutants, our data suggest that other GAP-associated proteins may also have roles in Src-mediated oncogenic transformation. These findings provide additional evidence for the specificity of Src interactions with GAP and support the hypothesis that these interactions contribute to the biological functions of the Scr kinases.
4
Brott, B. K., S. Decker, M. C. O'Brien, and R. Jove. "Molecular features of the viral and cellular Src kinases involved in interactions with the GTPase-activating protein." Molecular and Cellular Biology 11, no. 10 (October 1991): 5059–67. http://dx.doi.org/10.1128/mcb.11.10.5059.
Full textAbstract:
GTPase-activating protein (GAP) enhances the rate of GTP hydrolysis by cellular Ras proteins and is implicated in mitogenic signal transduction. GAP is phosphorylated on tyrosine in cells transformed by Rous sarcoma virus and serves as an in vitro substrate of the viral Src (v-Src) kinase. Our previous studies showed that GAP complexes stably with normal cellular Src (c-Src), although its association with v-Src is less stable. To further investigate the molecular basis for interactions between GAP and the Src kinases, we examined GAP association with and phosphorylation by a series of c-Src and v-Src mutants. Analysis of GAP association with c-Src/v-Src chimeric proteins demonstrates that GAP associates stably with Src proteins possessing low kinase activity and poorly with activated Src kinases, especially those that lack the carboxy-terminal segment of c-Src containing the regulatory amino acid Tyr-527. Phosphorylated Tyr-527 is a major determinant of c-Src association with GAP, as demonstrated by c-Src point mutants in which Tyr-527 is changed to Phe. While the isolated amino-terminal half of the c-Src protein is insufficient for stable GAP association, analysis of point substitutions of highly conserved amino acid residues in the c-Src SH2 region indicate that this region also influences Src-GAP complex formation. Therefore, our results suggest that both Tyr-527 phosphorylation and the SH2 region contribute to stable association of c-Src with GAP. Analysis of in vivo phosphorylation of GAP by v-Src mutants containing deletions encompassing the SH2, SH3, and unique regions suggests that the kinase domain of v-Src contains sufficient substrate specificity for GAP phosphorylation. Even though tyrosine phosphorylation of GAP correlates to certain extent with the transforming ability of various c-Src and v-Src mutants, our data suggest that other GAP-associated proteins may also have roles in Src-mediated oncogenic transformation. These findings provide additional evidence for the specificity of Src interactions with GAP and support the hypothesis that these interactions contribute to the biological functions of the Scr kinases.
5
Klomp, Jennifer E., Vincent Huyot, Anne-Marie Ray, Kerrie B. Collins, Asrar B. Malik, and Andrei V. Karginov. "Mimicking transient activation of protein kinases in living cells." Proceedings of the National Academy of Sciences 113, no. 52 (December 12, 2016): 14976–81. http://dx.doi.org/10.1073/pnas.1609675114.
Full textAbstract:
Physiological stimuli activate protein kinases for finite periods of time, which is critical for specific biological outcomes. Mimicking this transient biological activity of kinases is challenging due to the limitations of existing methods. Here, we report a strategy enabling transient kinase activation in living cells. Using two protein-engineering approaches, we achieve independent control of kinase activation and inactivation. We show successful regulation of tyrosine kinase c-Src (Src) and Ser/Thr kinase p38α (p38), demonstrating broad applicability of the method. By activating Src for finite periods of time, we reveal how the duration of kinase activation affects secondary morphological changes that follow transient Src activation. This approach highlights distinct roles for sequential Src-Rac1– and Src-PI3K–signaling pathways at different stages during transient Src activation. Finally, we demonstrate that this method enables transient activation of Src and p38 in a specific signaling complex, providing a tool for targeted regulation of individual signaling pathways.
6
Zhang, Jiayi, Ching-hang Wong, Weiliang Xia, Dolores D. Mruk, Nikki P. Y. Lee, Will M. Lee, and C. Yan Cheng. "Regulation of Sertoli-Germ Cell Adherens Junction Dynamics via Changes in Protein-Protein Interactions of the N-Cadherin-β-Catenin Protein Complex which Are Possibly Mediated by c-Src and Myotubularin-Related Protein 2: An in Vivo Study Using an Androgen Suppression Model." Endocrinology 146, no. 3 (March 1, 2005): 1268–84. http://dx.doi.org/10.1210/en.2004-1194.
Full textAbstract:
Using a well characterized model of cell-cell actin-based adherens junction (AJ) disruption by suppressing the intratesticular testosterone level in adult rats with testosterone-estradiol implants, we have confirmed earlier findings that Sertoli-germ cell AJ dynamics are regulated by the activation of kinases via putative signaling pathways but with some unexpected findings as follows. First, the loss of germ cells from the seminiferous epithelium during androgen suppression was associated with a surge in myotubularin-related protein 2 (MTMR2, a lipid phosphatase, in which adult MTMR2−/− mice were recently shown to be azoospermic because of the loss of cell adhesion function between germ and Sertoli cells); kinases: phosphatidylinositol 3-kinase, c-Src, and C-terminal Src kinase; adaptors: α-actinin, vinculin, afadin, and p130 Crk-associated protein; and AJ-integral membrane proteins at the ectoplasmic specialization (ES, a testis-specific cell-cell actin-based AJ type) site: N-cadherin, β-catenin, integrin β1, and nectin 3. Second, MTMR2, instead of structurally interacting with phosphatidylinositol 3-kinase, a protein and lipid kinase, was shown to associate only with c-Src, a nonreceptor protein tyrosine kinase, as demonstrated by both coimmunoprecipitation and fluorescent microscopy at the site of apical ES, but none of the kinases, adaptors, and AJ-integral proteins that were examined. Collectively, these results suggest that the MTMR2/c-Src is an important phosphatase/kinase protein pair in AJ dynamics in the testis. Because c-Src is known to associate with the cadherin/catenin protein complex at the ES in the testis, we next sought to investigate any changes in the protein-protein interactions of this protein complex during androgen suppression-induced germ cell loss. Indeed, there was a loss of N-cadherin and β-catenin association, accompanied by a surge in Tyr phosphorylation of β-catenin, during germ cell loss from the epithelium. Third, and perhaps the most important of all, during natural recovery of the epithelium after removal of testosterone-estradiol implants when spermatids were reattaching to Sertoli cells, an increase in N-cadherin and β-catenin association was detected with a concomitant loss in the increased Tyr phosphorylation in β-catenin. In summary, these results illustrate that the cadherin/catenin is a crucial cell adhesion complex that regulates AJ dynamics in the testis, and its functionality is likely modulated by the MTMR2/c-Src protein complex.
7
Richard, S., D. Yu, K. J. Blumer, D. Hausladen, M. W. Olszowy, P. A. Connelly, and A. S. Shaw. "Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1." Molecular and Cellular Biology 15, no. 1 (January 1995): 186–97. http://dx.doi.org/10.1128/mcb.15.1.186.
Full textAbstract:
src family tyrosine kinases contain two noncatalytic domains termed src homology 3 (SH3) and SH2 domains. Although several other signal transduction molecules also contain tandemly occurring SH3 and SH2 domains, the function of these closely spaced domains is not well understood. To identify the role of the SH3 domains of src family tyrosine kinases, we sought to identify proteins that interacted with this domain. By using the yeast two-hybrid system, we identified p62, a tyrosine-phosphorylated protein that associates with p21ras GTPase-activating protein, as a src family kinase SH3-domain-binding protein. Reconstitution of complexes containing p62 and the src family kinase p59fyn in HeLa cells demonstrated that complex formation resulted in tyrosine phosphorylation of p62 and was mediated by both the SH3 and SH2 domains of p59fyn. The phosphorylation of p62 by p59fyn required an intact SH3 domain, demonstrating that one function of the src family kinase SH3 domains is to bind and present certain substrates to the kinase. As p62 contains at least five SH3-domain-binding motifs and multiple tyrosine phosphorylation sites, p62 may interact with other signalling molecules via SH3 and SH2 domain interactions. Here we show that the SH3 and/or SH2 domains of the signalling proteins Grb2 and phospholipase C gamma-1 can interact with p62 both in vitro and in vivo. Thus, we propose that one function of the tandemly occurring SH3 and SH2 domains of src family kinases is to bind p62, a multifunctional SH3 and SH2 domain adapter protein, linking src family kinases to downstream effector and regulatory molecules.
8
Ahn, Bong-Hyun, Shi Yeon Kim, Eun Hee Kim, Kyeong Sook Choi, Taeg Kyu Kwon, Young Han Lee, Jong-Soo Chang, Myung-Suk Kim, Yang-Hyeok Jo, and Do Sik Min. "Transmodulation between Phospholipase D and c-Src Enhances Cell Proliferation." Molecular and Cellular Biology 23, no. 9 (May 1, 2003): 3103–15. http://dx.doi.org/10.1128/mcb.23.9.3103-3115.2003.
Full textAbstract:
ABSTRACT Phospholipase D (PLD) has been implicated in the signal transduction pathways initiated by several mitogenic protein tyrosine kinases. We demonstrate for the first time that most notably PLD2 and to a lesser extent the PLD1 isoform are tyrosine phosphorylated by c-Src tyrosine kinase via direct association. Moreover, epidermal growth factor induced tyrosine phosphorylation of PLD2 and its interaction with c-Src in A431 cells. Interaction between these proteins is via the pleckstrin homology domain of PLD2 and the catalytic domain of c-Src. Coexpression of PLD1 or PLD2 with c-Src synergistically enhances cellular proliferation compared with expression of either molecule. While PLD activity as a lipid-hydrolyzing enzyme is not affected by c-Src, wild-type PLDs but not catalytically inactive PLD mutants significantly increase c-Src kinase activity, up-regulating c-Src-mediated paxillin phosphorylation and extracellular signal-regulated kinase activity. These results demonstrate the critical role of PLD catalytic activity in the stimulation of Src signaling. In conclusion, we provide the first evidence that c-Src acts as a kinase of PLD and PLD acts as an activator of c-Src. This transmodulation between c-Src and PLD may contribute to the promotion of cellular proliferation via amplification of mitogenic signaling pathways.
9
Wang, Y. H., F. Li, J. H. Schwartz, P. J. Flint, and S. C. Borkan. "c-Src and HSP72 interact in ATP-depleted renal epithelial cells." American Journal of Physiology-Cell Physiology 281, no. 5 (November 1, 2001): C1667—C1675. http://dx.doi.org/10.1152/ajpcell.2001.281.5.c1667.
Full textAbstract:
Disruption of cell contact sites during ischemia contributes to the loss of organ function in acute renal failure. Because prior heat stress protects cell contact sites in ATP-depleted renal epithelial cells in vitro, we hypothesized that heat shock protein 72 (HSP72), the major inducible cytoprotectant in mammalian cells, interacts with protein kinases that regulate cell-cell and cell-matrix interactions. ATP depletion increased the content of Tyr416 Src, the activated form of this kinase. c-Src activation was associated with an increase in the tyrosine phosphorylation state of β-catenin, paxillin, and vinculin, three c-Src substrate proteins that localize to and regulate cell contact sites. Prior heat stress inhibited c-Src activation and decreased the degree of tyrosine phosphorylation of all three Src substrates during ATP depletion and/or early recovery. HSP72 coimmunoprecipitated with c-Src only in cells subjected to heat stress. ATP depletion markedly increased the interaction between HSP72 and c-Src, supporting the hypothesis that HSP72 regulates Src kinase activity. These results suggest that alterations in the tyrosine phosphorylation state of proteins located at the cell-cell and cell-matrix interface mediate, at least in part, the functional state of these structures during ATP depletion and may be modulated by interactions between HSP72 and c-Src.
10
Levy, J. B., and J. S. Brugge. "Biological and biochemical properties of the c-src+ gene product overexpressed in chicken embryo fibroblasts." Molecular and Cellular Biology 9, no. 8 (August 1989): 3332–41. http://dx.doi.org/10.1128/mcb.9.8.3332-3341.1989.
Full textAbstract:
The c-src protein isolated from neuronal cells (pp60c-src+) displays a higher level of protein kinase activity than does pp60c-src from nonneural tissues. There are two structural alterations present in the amino-terminal half of pp60c-src+ expressed in neurons which could contribute to the enhanced activity of this form of pp60c-src: (i) a hexapeptide insert located at amino acid 114 of avian pp60c-src+ and (ii) a novel site(s) of serine phosphorylation. We characterized pp60c-src+ expressed in a nonneuronal cell type to identify factors that regulate the activity of the c-src+ protein and the importance of the neuronal environment on this regulation. The c-src+ protein overexpressed in chicken embryo fibroblasts (CEFs) displayed higher kinase activity than did pp60c-src. The major sites of phosphorylation of the c-src+ protein were Ser-17 and Tyr-527. The unique site(s) of serine phosphorylation originally identified in pp60c-src+ expressed in neurons was not detected in the c-src+ protein overexpressed in CEFs. Therefore, the hexapeptide insert is sufficient to cause an elevation in the tyrosine protein kinase activity of pp60c-src+. Our data also indicate that CEFs infected with the Rous sarcoma virus (RSV)c-src+ display phenotypic changes that distinguish them from cultures producing pp60c-src and that pp60c-src+-expressing cells are better able to grow in an anchorage-independent manner. The level of total cellular tyrosine phosphorylation in RSVc-src+-infected cultures was moderately higher than the level observed in cultures infected with RSVc-src. This level was not as pronounced as that observed in cells infected with RSVv-src or oncogenic variants of RSVc-src. Thus, pp60c-src+ could be considered a partially activated c-src variant protein much like other c-src proteins that contain mutations in the amino-terminal domain.
11
Levy, J. B., and J. S. Brugge. "Biological and biochemical properties of the c-src+ gene product overexpressed in chicken embryo fibroblasts." Molecular and Cellular Biology 9, no. 8 (August 1989): 3332–41. http://dx.doi.org/10.1128/mcb.9.8.3332.
Full textAbstract:
The c-src protein isolated from neuronal cells (pp60c-src+) displays a higher level of protein kinase activity than does pp60c-src from nonneural tissues. There are two structural alterations present in the amino-terminal half of pp60c-src+ expressed in neurons which could contribute to the enhanced activity of this form of pp60c-src: (i) a hexapeptide insert located at amino acid 114 of avian pp60c-src+ and (ii) a novel site(s) of serine phosphorylation. We characterized pp60c-src+ expressed in a nonneuronal cell type to identify factors that regulate the activity of the c-src+ protein and the importance of the neuronal environment on this regulation. The c-src+ protein overexpressed in chicken embryo fibroblasts (CEFs) displayed higher kinase activity than did pp60c-src. The major sites of phosphorylation of the c-src+ protein were Ser-17 and Tyr-527. The unique site(s) of serine phosphorylation originally identified in pp60c-src+ expressed in neurons was not detected in the c-src+ protein overexpressed in CEFs. Therefore, the hexapeptide insert is sufficient to cause an elevation in the tyrosine protein kinase activity of pp60c-src+. Our data also indicate that CEFs infected with the Rous sarcoma virus (RSV)c-src+ display phenotypic changes that distinguish them from cultures producing pp60c-src and that pp60c-src+-expressing cells are better able to grow in an anchorage-independent manner. The level of total cellular tyrosine phosphorylation in RSVc-src+-infected cultures was moderately higher than the level observed in cultures infected with RSVc-src. This level was not as pronounced as that observed in cells infected with RSVv-src or oncogenic variants of RSVc-src. Thus, pp60c-src+ could be considered a partially activated c-src variant protein much like other c-src proteins that contain mutations in the amino-terminal domain.
12
Burnham, Mary Rose, Pamela J. Bruce-Staskal, Mary T. Harte, Cheryl L. Weidow, Amy Ma, Scott A. Weed, and Amy H. Bouton. "Regulation of c-SRC Activity and Function by the Adapter Protein CAS." Molecular and Cellular Biology 20, no. 16 (August 15, 2000): 5865–78. http://dx.doi.org/10.1128/mcb.20.16.5865-5878.2000.
Full textAbstract:
ABSTRACT SRC family kinases play essential roles in a variety of cellular functions, including proliferation, survival, differentiation, and apoptosis. The activities of these kinases are regulated by intramolecular interactions and by heterologous binding partners that modulate the transition between active and inactive structural conformations. p130 CAS (CAS) binds directly to both the SH2 and SH3 domains of c-SRC and therefore has the potential to structurally alter and activate this kinase. In this report, we demonstrate that overexpression of full-length CAS in COS-1 cells induces c-SRC-dependent tyrosine phosphorylation of multiple endogenous cellular proteins. A carboxy-terminal fragment of CAS (CAS-CT), which contains the c-SRC binding site, was sufficient to induce c-SRC-dependent protein tyrosine kinase activity, as measured by tyrosine phosphorylation of cortactin, paxillin, and, to a lesser extent, focal adhesion kinase. A single amino acid substitution located in the binding site for the SRC SH3 domain of CAS-CT disrupted CAS-CT's interaction with c-SRC and inhibited its ability to induce tyrosine phosphorylation of cortactin and paxillin. Murine C3H10T1/2 fibroblasts that expressed elevated levels of tyrosine phosphorylated CAS and c-SRC–CAS complexes exhibited an enhanced ability to form colonies in soft agar and to proliferate in the absence of serum or growth factors. CAS-CT fully substituted for CAS in mediating growth in soft agar but was less effective in promoting serum-independent growth. These data suggest that CAS plays an important role in regulating specific signaling pathways governing cell growth and/or survival, in part through its ability to interact with and modulate the activity of c-SRC.
13
Yan, S. R., M. Huang, and G. Berton. "Signaling by adhesion in human neutrophils: activation of the p72syk tyrosine kinase and formation of protein complexes containing p72syk and Src family kinases in neutrophils spreading over fibrinogen." Journal of Immunology 158, no. 4 (February 15, 1997): 1902–10. http://dx.doi.org/10.4049/jimmunol.158.4.1902.
Full textAbstract:
Abstract Src family kinases are implicated in signaling by integrins in polymorphonuclear neutrophils (PMN). To identify proteins present in complexes with Src family kinases, we subjected p58(c-fgr) or p53/56(lyn) immunoprecipitates from Triton X-100 lysates of PMN incubated on fibrinogen-coated surfaces to in vitro kinase assays. Assays on p58(c-fgr) or p53/56(lyn) immunoprecipitates from Triton lysates of PMN induced to spread over fibrinogen in response to TNF-alpha showed that several proteins form complexes with Src family kinases and can be phosphorylated in vitro. Immunoblot analysis showed that the p72(syk) tyrosine kinase is one of these proteins. Formation of protein complexes containing Src family kinases and p72(syk) required PMN spreading because p72(syk) was not detected in p58(c-fgr) or p53/56(lyn) immunoprecipitates from PMN, which were stimulated with TNF-alpha, in suspension. In addition, induction of PMN spreading with Mn2+ in the absence of TNF-alpha promoted the formation of protein complexes containing Src family kinases and p72(syk). We also found that p72(syk)-autophosphorylating kinase activity was enhanced, and a fraction of p72(syk) was translocated to a Triton-insoluble cytoskeletal fraction in PMN induced to spread over fibrinogen by TNF-alpha or Mn2+. In vitro kinase assays on CD18 (beta 2 integrin subunit) immunoprecipitates from Triton lysates of spread PMN showed that several proteins formed complexes with CD18 and could be phosphorylated in vitro. Immunoblot analysis of CD18 immunoprecipitates allowed us to identify p72(syk) as one of these proteins. These findings show that PMN spreading is accompanied by activation of p72(syk) and formation of multimolecular complexes in which Src family kinases and p72(syk) colocalize.
14
Patwardhan, Parag, and Marilyn D. Resh. "Myristoylation and Membrane Binding Regulate c-Src Stability and Kinase Activity." Molecular and Cellular Biology 30, no. 17 (June 28, 2010): 4094–107. http://dx.doi.org/10.1128/mcb.00246-10.
Full textAbstract:
ABSTRACT Myristoylation is critical for membrane association of Src kinases, but a role for myristate in regulating other aspects of Src biology has not been explored. In the c-Abl tyrosine kinase, myristate binds within a hydrophobic pocket at the base of the kinase domain and latches the protein into an autoinhibitory conformation. A similar pocket has been predicted to exist in c-Src, raising the possibility that Src might also be regulated by myristoylation. Here we show that in contrast to the case for c-Abl, myristoylation exerts a positive effect on c-Src kinase activity. We also demonstrate that myristoylation and membrane binding regulate c-Src ubiquitination and degradation. Nonmyristoylated c-Src exhibited reduced kinase activity but had enhanced stability compared to myristoylated c-Src. We then mutated critical residues in the predicted myristate binding pocket of c-Src. Mutation of L360 and/or E486 had no effect on c-Src membrane binding or localization. However, constructs containing a T456A mutation were partially released from the membrane, suggesting that mutagenesis could induce c-Src to undergo an artificial myristoyl switch. All of the pocket mutants exhibited decreased kinase activity. We concluded that myristoylation and the pocket residues regulate c-Src, but in a manner very different from that for c-Abl.
15
Nemeth, S. P., L. G. Fox, M. DeMarco, and J. S. Brugge. "Deletions within the amino-terminal half of the c-src gene product that alter the functional activity of the protein." Molecular and Cellular Biology 9, no. 3 (March 1989): 1109–19. http://dx.doi.org/10.1128/mcb.9.3.1109-1119.1989.
Full textAbstract:
To examine how amino acid sequences outside of the catalytic domain of pp60c-src influence the functional activity of this protein, we have introduced deletion mutations within the amino-terminal half of pp60c-src. These mutations caused distinct changes in the biochemical properties of the c-src gene products and in the properties of cells infected with retroviruses carrying these mutant c-src genes. Cells expressing the c-srcNX protein, which contains a deletion of amino acids 15 to 89, displayed a refractile, spindle-shaped morphology, formed intermediate-sized, tightly packed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Thus, deletion of amino acids 15 to 89 can activate the kinase activity and transforming potential of the c-src gene product. Deletion of amino acids 112 to 225, however, did not increase the kinase activity or transforming ability of pp60c-src; indeed, deletion of these sequences in c-srcHP suppressed phenotypic alterations induced by pp60c-src. Cells expressing the c-srcNP or c-srcBS gene products (containing deletions of amino acids 15 to 225 and 55 to 169, respectively) displayed a fusiform, refractile morphology and formed diffuse colonies in soft agar; the mutant proteins displayed an increased in vitro protein-tyrosine kinase activity. However, only a few cellular proteins contained elevated levels of phosphotyrosine in vivo. Thus, deletions downstream of amino acid 89 severely restricted the ability of c-src to phosphorylate cellular substrates in vivo without affecting the intrinsic tyrosine kinase activity of the c-src gene product. These results suggest the existence of at least two modulatory regions within the amino-terminal half of pp60c-src that are important for the regulation of tyrosine kinase activity and for the interaction of pp60c-src with cellular substrates.
16
Nemeth, S. P., L. G. Fox, M. DeMarco, and J. S. Brugge. "Deletions within the amino-terminal half of the c-src gene product that alter the functional activity of the protein." Molecular and Cellular Biology 9, no. 3 (March 1989): 1109–19. http://dx.doi.org/10.1128/mcb.9.3.1109.
Full textAbstract:
To examine how amino acid sequences outside of the catalytic domain of pp60c-src influence the functional activity of this protein, we have introduced deletion mutations within the amino-terminal half of pp60c-src. These mutations caused distinct changes in the biochemical properties of the c-src gene products and in the properties of cells infected with retroviruses carrying these mutant c-src genes. Cells expressing the c-srcNX protein, which contains a deletion of amino acids 15 to 89, displayed a refractile, spindle-shaped morphology, formed intermediate-sized, tightly packed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Thus, deletion of amino acids 15 to 89 can activate the kinase activity and transforming potential of the c-src gene product. Deletion of amino acids 112 to 225, however, did not increase the kinase activity or transforming ability of pp60c-src; indeed, deletion of these sequences in c-srcHP suppressed phenotypic alterations induced by pp60c-src. Cells expressing the c-srcNP or c-srcBS gene products (containing deletions of amino acids 15 to 225 and 55 to 169, respectively) displayed a fusiform, refractile morphology and formed diffuse colonies in soft agar; the mutant proteins displayed an increased in vitro protein-tyrosine kinase activity. However, only a few cellular proteins contained elevated levels of phosphotyrosine in vivo. Thus, deletions downstream of amino acid 89 severely restricted the ability of c-src to phosphorylate cellular substrates in vivo without affecting the intrinsic tyrosine kinase activity of the c-src gene product. These results suggest the existence of at least two modulatory regions within the amino-terminal half of pp60c-src that are important for the regulation of tyrosine kinase activity and for the interaction of pp60c-src with cellular substrates.
17
Tobe, K., H. Sabe, T. Yamamoto, T. Yamauchi, S. Asai, Y. Kaburagi, H. Tamemoto, et al. "Csk enhances insulin-stimulated dephosphorylation of focal adhesion proteins." Molecular and Cellular Biology 16, no. 9 (September 1996): 4765–72. http://dx.doi.org/10.1128/mcb.16.9.4765.
Full textAbstract:
Insulin has pleiotropic effects on the regulation of cell physiology through binding to its receptor. The wide variety of tyrosine phosphorylation motifs of insulin receptor substrate 1 (IRS-1), a substrate for the activated insulin receptor tyrosine kinase, may account for the multiple functions of insulin. Recent studies have shown that activation of the insulin receptor leads to the regulation of focal adhesion proteins, such as a dephosphorylation of focal adhesion kinase (pp125FAK). We show here that C-terminal Src kinase (Csk), which phosphorylates C-terminal tyrosine residues of Src family protein tyrosine kinases and suppresses their kinase activities, is involved in this insulin-stimulated dephosphorylation of focal adhesion proteins. We demonstrated that the overexpression of Csk enhanced and prolonged the insulin-induced dephosphorylation of pp125FAK. Another focal adhesion protein, paxillin, was also dephosphorylated upon insulin stimulation, and a kinase-negative mutant of Csk was able to inhibit the insulin-induced dephosphorylation of pp125FAK and paxillin. Although we have shown that the Csk Src homology 2 domain can bind to several tyrosine-phosphorylated proteins, including pp125FAK and paxillin, a majority of protein which bound to Csk was IRS-1 when cells were stimulated by insulin. Our data also indicated that tyrosine phosphorylation levels of IRS-1 appear to be paralleled by the dephosphorylation of the focal adhesion proteins. We therefore propose that the kinase activity of Csk, through the insulin-induced complex formation of Csk with IRS-1, is involved in insulin's regulation of the phosphorylation levels of the focal adhesion proteins, possibly through inactivation of the kinase activity of c-Src family kinases.
18
Zhao, Y., H. Uyttendaele, J. G. Krueger, M. Sudol, and H. Hanafusa. "Inactivation of c-Yes tyrosine kinase by elevation of intracellular calcium levels." Molecular and Cellular Biology 13, no. 12 (December 1993): 7507–14. http://dx.doi.org/10.1128/mcb.13.12.7507-7514.1993.
Full textAbstract:
We have previously shown that the c-Src tyrosine kinase is activated four- to fivefold when cultured keratinocytes differentiate following the elevation of intracellular calcium levels. In contrast to c-Src, another Src family tyrosine kinase, c-Yes, was rapidly inactivated in these same cells, despite its marked similarity in structure and enzymatic activity to c-Src. The inactivation of c-Yes was independent of the protein kinase C pathway, which is usually activated by elevation of intracellular calcium levels. The protein levels of c-Src and c-Yes were not altered, but the phosphotyrosine content of both proteins was greatly reduced. As has been demonstrated for c-Src, in vitro dephosphorylation of c-Yes by incubation with protein tyrosine phosphatases also resulted in its activation, not inactivation. In vitro reconstitution experiments showed that c-Yes can be inactivated by preincubation with a Ca(2+)-supplemented cell extract and that this inhibition was reversed by the addition of EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. Gradient sedimentation of cell lysates showed that in cells treated with calcium and ionophore, c-Yes formed complexes with two distinct cellular proteins, whereas similar complexes were not seen in c-Src immunoprecipitates. One of these two proteins has the ability to inhibit c-Yes kinase activity in vitro. Finally, the Ca(2+)-dependent inactivation of c-Yes was observed in kidney tubular cells and fibroblasts, suggesting that the Ca(2+)-dependent regulation of c-Yes tyrosine kinase is not unique to keratinocytes. We postulate that c-Yes is inactivated through a Ca2+ -dependent association with cellular proteins, which seems to override its activation resulting from tyrosine dephosphorylation.
19
Zhao, Y., H. Uyttendaele, J. G. Krueger, M. Sudol, and H. Hanafusa. "Inactivation of c-Yes tyrosine kinase by elevation of intracellular calcium levels." Molecular and Cellular Biology 13, no. 12 (December 1993): 7507–14. http://dx.doi.org/10.1128/mcb.13.12.7507.
Full textAbstract:
We have previously shown that the c-Src tyrosine kinase is activated four- to fivefold when cultured keratinocytes differentiate following the elevation of intracellular calcium levels. In contrast to c-Src, another Src family tyrosine kinase, c-Yes, was rapidly inactivated in these same cells, despite its marked similarity in structure and enzymatic activity to c-Src. The inactivation of c-Yes was independent of the protein kinase C pathway, which is usually activated by elevation of intracellular calcium levels. The protein levels of c-Src and c-Yes were not altered, but the phosphotyrosine content of both proteins was greatly reduced. As has been demonstrated for c-Src, in vitro dephosphorylation of c-Yes by incubation with protein tyrosine phosphatases also resulted in its activation, not inactivation. In vitro reconstitution experiments showed that c-Yes can be inactivated by preincubation with a Ca(2+)-supplemented cell extract and that this inhibition was reversed by the addition of EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. Gradient sedimentation of cell lysates showed that in cells treated with calcium and ionophore, c-Yes formed complexes with two distinct cellular proteins, whereas similar complexes were not seen in c-Src immunoprecipitates. One of these two proteins has the ability to inhibit c-Yes kinase activity in vitro. Finally, the Ca(2+)-dependent inactivation of c-Yes was observed in kidney tubular cells and fibroblasts, suggesting that the Ca(2+)-dependent regulation of c-Yes tyrosine kinase is not unique to keratinocytes. We postulate that c-Yes is inactivated through a Ca2+ -dependent association with cellular proteins, which seems to override its activation resulting from tyrosine dephosphorylation.
20
Adolph, Dörte, Nadine Flach, Katharina Mueller, Dirk H. Ostareck, and Antje Ostareck-Lederer. "Deciphering the Cross Talk between hnRNP K and c-Src: the c-Src Activation Domain in hnRNP K Is Distinct from a Second Interaction Site." Molecular and Cellular Biology 27, no. 5 (December 18, 2006): 1758–70. http://dx.doi.org/10.1128/mcb.02014-06.
Full textAbstract:
ABSTRACT The protein tyrosine kinase c-Src is regulated by two intramolecular interactions. The repressed state is achieved through the interaction of the Src homology 2 (SH2) domain with the phosphorylated C-terminal tail and the association of the SH3 domain with a polyproline type II helix formed by the linker region between SH2 and the kinase domain. hnRNP K, the founding member of the KH domain protein family, is involved in chromatin remodeling, regulation of transcription, and translation of specific mRNAs and is a target in different signal transduction pathways. In particular, it functions as a specific activator and a substrate of the tyrosine kinase c-Src. Here we address the question how hnRNP K interacts with and activates c-Src. We define the proline residues in hnRNP K in the proline-rich motifs P2 (amino acids [aa] 285 to 297) and P3 (aa 303 to 318), which are necessary and sufficient for the specific activation of c-Src, and we dissect the amino acid sequence (aa 216 to 226) of hnRNP K that mediates a second interaction with c-Src. Our findings indicate that the interaction with c-Src and the activation of the kinase are separable functions of hnRNP K. hnRNP K acts as a scaffold protein that integrates signaling cascades by facilitating the cross talk between kinases and factors that mediate nucleic acid-directed processes.
21
Mukhopadhyay, Chandrani, Aleata Triplett, Tom Bargar, Carol Heckman, Kay-Uwe Wagner, and Mayumi Naramura. "Casitas B-cell lymphoma (Cbl) proteins protect mammary epithelial cells from proteotoxicity of active c-Src accumulation." Proceedings of the National Academy of Sciences 113, no. 51 (December 5, 2016): E8228—E8237. http://dx.doi.org/10.1073/pnas.1615677113.
Full textAbstract:
Casitas B-cell lymphoma (Cbl) family ubiquitin ligases negatively regulate tyrosine kinase-dependent signal transduction by promoting degradation of active kinases. We and others previously reported that loss of Cbl functions caused hyperproliferation in lymphoid and hematopoietic systems. Unexpectedly, Cbl deletion in Cbl-b–null, Cbl-c–null primary mouse mammary epithelial cells (MECs) (Cbl triple-deficiency) induced rapid cell death despite enhanced MAP kinase and AKT activation. Acute Cbl triple-deficiency elicited distinct transcriptional and biochemical responses with partial overlap with previously described cellular reactions to unfolded proteins and oxidative stress. Although the levels of reactive oxygen species were comparable, detergent-insoluble protein aggregates containing phosphorylated c-Src accumulated in Cbl triple-deficient MECs. Treatment with a broad-spectrum kinase inhibitor dasatinib blocked protein aggregate accumulation and restored in vitro organoid formation. This effect is most likely mediated through c-Src because Cbl triple-deficient MECs were able to form organoids upon shRNA-mediated c-Src knockdown. Taking these data together, the present study demonstrates that Cbl family proteins are required to protect MECs from proteotoxic stress-induced cell death by promoting turnover of active c-Src.
22
Cunningham-Smith, K. A., R. Witherdin, L. Hetherington, M. A. Baker, and R. Aitken. "277.The role of pp60c-src tyrosine kinase in sperm capacitation." Reproduction, Fertility and Development 16, no. 9 (2004): 277. http://dx.doi.org/10.1071/srb04abs277.
Full textAbstract:
A protein kinase A-dependent tyrosine phosphorylation pathway in mammalian spermatozoa has been demonstrated to exist, and is unique to this cell type. As PKA is incapable of directly phosphorylating substrates on tyrosine residues, much research has focused on the identification of an intermediary tyrosine kinase which can be activated by serine/threonine phosphorylation via PKA. Inhibitory studies using genistein, tryphostin, erbstatin and herbimycin A, have demonstrated that the src family of kinases may be responsible for the tyrosine phosphorylation events seen during capacitation (1). Although one src family member, c-yes, has been implicated as the kinase responsible for these events (2), this has since been disputed (3). Another src family member, pp60c-src, can be activated by phosphorylation on Ser-17 by cAMP-dependent protein kinase and Ser-12 by calcium-phospholipid dependent protein kinase C (4), and may be the intermediary tyrosine kinase of interest. Western blot analysis demonstrated the presence of pp60c-src in rat sperm samples isolated from the caput and cauda epididymis. Furthermore, co-immunoprecipitation studies revealed a number of pp60c-src-associated proteins including outer dense fibre 2 (ODF2) and A-kinase anchoring protein 4 (AKAP4). Interestingly, both of these proteins become phosphorylated during capacitation of mouse sperm (data not shown) and AKAP4 is tyrosine phosphorylated in capacitated human sperm (5). These data implicate pp60c-src kinase activity in the phosphorylation of a number of sperm midpiece proteins, which may regulate hyperactivation during capacitation. Further research focusing on the activity of pp60c-src in non-capacitated and capacitated sperm will be conducted. (1) Yaciuk P., et al. (1989) Mol. Cell Biol. 9, 2453–2461. (2) Baker M., et al. (2003) J. Cell Sci. 117, 211–222. (3) Leclerc P., Goupil S (2002) Biol. Reprod. 67, 301–307. (4) Bajpai M., et al. (2003) Arch. Androl. 49, 229–246. (5) Ficarro S., et al. (2003) J. Biol. Chem. 278, 11�579–11�589.
23
Amatya, Neha, David Yin-wei Lin, and Amy H. Andreotti. "Dynamic regulatory features of the protein tyrosine kinases." Biochemical Society Transactions 47, no. 4 (August 8, 2019): 1101–16. http://dx.doi.org/10.1042/bst20180590.
Full textAbstract:
Abstract The SRC, Abelson murine leukemia viral oncogene homolog 1, TEC and C-terminal SRC Kinase families of non-receptor tyrosine kinases (collectively the Src module kinases) mediate an array of cellular signaling processes and are therapeutic targets in many disease states. Crystal structures of Src modules kinases provide valuable insights into the regulatory mechanisms that control activation and generate a framework from which drug discovery can advance. The conformational ensembles visited by these multidomain kinases in solution are also key features of the regulatory machinery controlling catalytic activity. Measurement of dynamic motions within kinases substantially augments information derived from crystal structures. In this review, we focus on a body of work that has transformed our understanding of non-receptor tyrosine kinase regulation from a static view to one that incorporates how fluctuations in conformational ensembles and dynamic motions influence activation status. Regulatory dynamic networks are often shared across and between kinase families while specific dynamic behavior distinguishes unique regulatory mechanisms for select kinases. Moreover, intrinsically dynamic regions of kinases likely play important regulatory roles that have only been partially explored. Since there is clear precedence that kinase inhibitors can exploit specific dynamic features, continued efforts to define conformational ensembles and dynamic allostery will be key to combating drug resistance and devising alternate treatments for kinase-associated diseases.
24
Yin, Guoyong, Judith Haendeler, Chen Yan, and Bradford C. Berk. "GIT1 Functions as a Scaffold for MEK1-Extracellular Signal-Regulated Kinase 1 and 2 Activation by Angiotensin II and Epidermal Growth Factor." Molecular and Cellular Biology 24, no. 2 (January 15, 2004): 875–85. http://dx.doi.org/10.1128/mcb.24.2.875-885.2004.
Full textAbstract:
ABSTRACT Activation of the mitogen-activated protein kinase pathway represented by extracellular signal-regulated kinases (ERK1/2) and activation of the upstream kinase (MEK1) are critical events for growth factor signal transduction. c-Src has been proposed as a common mediator for these signals in response to both G protein-coupled receptors (GPCRs) and tyrosine kinase-coupled receptors (TKRs). Here we show that the GPCR kinase-interacting protein 1 (GIT1) is a substrate for c-Src that associates with MEK1 in vascular smooth-muscle cells and human embryonic kidney 293 cells. GIT1 binding via coiled-coil domains and a Spa2 homology domain is required for sustained activation of MEK1-ERK1/2 after stimulation with angiotensin II and epidermal growth factor. We propose that GIT1 serves as a scaffold protein to facilitate c-Src-dependent activation of MEK1-ERK1/2 in response to both GPCRs and TKRs.
25
Salinas-Garcia, M. Carmen, Marina Plaza-Garrido, and Ana Camara-Artigas. "The impact of oncogenic mutations of the viral Src kinase on the structure and stability of the SH3 domain." Acta Crystallographica Section D Structural Biology 77, no. 6 (May 19, 2021): 854–66. http://dx.doi.org/10.1107/s2059798321004344.
Full textAbstract:
Src kinase belongs to the family of Src-related nonreceptor tyrosine kinases. Because of its physiological role in cell growth and proliferation, its activity is strictly controlled by several mechanisms. Nevertheless, in viral Src kinase (v-Src) some of these mechanisms fail, and its uncontrolled activity is responsible for the occurrence of cancer. Here, the crystal structures of three SH3-domain mutants of v-Src were determined to unveil the effects of these oncogenic mutations in this regulatory domain. Mutations in the n-Src and distal loops have a low impact on the overall structure of the domain and its capacity to form intertwined dimers. However, mutations in the RT loop compromise the stability of the domain and make the protein very prone to aggregation. Additionally, these mutations prevent the formation of intertwined dimers. The results show a synergistic effect between mutations in the RT loop and those in the n-Src and distal loops. Analysis of the structures of the v-Src SH3-domain mutants and the closed inactive conformation of cellular Src kinase (c-Src) point to a loss of the interactions that are required to establish the compact inactive form of the kinase. Nevertheless, an analysis of structures of the c-Src SH3 domain complexed with class I and II peptides points to minor changes in the interactions between the v-Src SH3 domain and these peptides. In this way, the structures reported here indicate that mutations in the RT loop might impair the kinase regulation mechanism without affecting the recognition of short proline-rich motifs in the target proteins of the kinase, thus explaining the oncogenic behaviour of the protein.
26
Boschelli, F., S. M. Uptain, and J. J. Lightbody. "The lethality of p60v-src in Saccharomyces cerevisiae and the activation of p34CDC28 kinase are dependent on the integrity of the SH2 domain." Journal of Cell Science 105, no. 2 (June 1, 1993): 519–28. http://dx.doi.org/10.1242/jcs.105.2.519.
Full textAbstract:
The lethal effects of the expression of the oncogenic protein tyrosine kinase p60v-src in Saccharomyces cerevisiae are associated with a loss of cell cycle control at the G1/S and G2/M checkpoints. Results described here indicate that the ability of v-Src to kill yeast is dependent on the integrity of the SH2 domain, a region of the Src protein involved in recognition of proteins phosphorylated on tyrosine. Catalytically active v-Src proteins with deletions in the SH2 domain have little effect on yeast growth, unlike wild-type v-Src protein, which causes accumulation of large-budded cells, perturbation of spindle microtubules and increased DNA content when expressed. The proteins phosphorylated on tyrosine in cells expressing v-Src differ from those in cells expressing a Src protein with a deletion in the SH2 domain. Also, unlike the wild-type v-Src protein, which drastically increases histone H1-associated Cdc28 kinase activity, c-Src and an altered v-Src protein have no effect on Cdc28 kinase activity. These results indicate that the SH2 domain is functionally important in the disruption of the yeast cell cycle by v-Src.
27
Gentili, Claudia, Ricardo Boland, and Ana Russo de Boland. "Implication of Gβγ proteins and c-SRC tyrosine kinase in parathyroid hormone-induced signal transduction in rat enterocytes." Journal of Endocrinology 188, no. 1 (January 2006): 69–78. http://dx.doi.org/10.1677/joe.1.06397.
Full textAbstract:
Parathyroid hormone (PTH) interacts in target tissues with a G protein-coupled receptor (GPCR) localized in the plasma membrane. Although activation of GPCR can elicit rapid stimulation of cellular protein tyrosine phosphorylation, the mechanism by which G proteins activate protein-tyrosine kinases is not completely understood. In the present work, we demonstrate that PTH rapidly increases the activity of non-receptor tyrosine kinase c-Src in rat intestinal cells (enterocytes). The response is biphasic, the early phase is fast and transient, peaking at 30 s (+120%), while the second phase progressively increases up to 5 min (+220%). The hormone activates c-Src in intestinal cells through fast changes in tyrosine phosphorylation of the enzyme. The first event in the activation of c-Src is the dephosphorylation of Tyr527 (which happens after a few seconds of PTH treatment), followed by a second event of activation with phosphorylation at Tyr416 (+twofold, 5 min). Removal of external Ca2+ (EGTA, 0.5 mM) and chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetracetic acid acetoxymethyl ester (BAPTA) (5 μM) suppressed Tyr527 dephosphorylation and Tyr416 phosphorylation, indicating that Ca2+ is an upstream activator of c-Src in enterocytes stimulated with PTH. The G protein subunits, Gαs and Gβ, are associated with c-Src in basal conditions and this association increases two- to threefold in cells treated with PTH. Blocking of Gβ subunits by preincubation of cells with a Gβ antibody abolished hormone-dependent c-Src Tyr416 phosphorylation and ERK1/ERK2 activation. The results of this work indicate that PTH activates c-Src in intestinal cells through conformational changes via G proteins and calcium-dependent modulation of tyrosine phosphorylation of the enzyme, and that PTH receptor activation leads via Gβγ–c-Src to the phosphorylation of the MAP kinases, ERK1 and ERK2.
28
Chong, Yuh-Ping, Terrence D. Mulhern, and Heung-Chin Cheng. "C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK)—endogenous negative regulators of Src-family protein kinases." Growth Factors 23, no. 3 (January 2005): 233–44. http://dx.doi.org/10.1080/08977190500178877.
Full text
29
Place, Aaron T., Zhenlong Chen, Farnaz R. Bakhshi, Guoquan Liu, John P. O'Bryan, and Richard D. Minshall. "Cooperative Role of Caveolin-1 and C-Terminal Src Kinase Binding Protein in C-Terminal Src Kinase-Mediated Negative Regulation of c-Src." Molecular Pharmacology 80, no. 4 (July 21, 2011): 665–72. http://dx.doi.org/10.1124/mol.111.073957.
Full text
30
Shvartsman, Dmitry E., John C. Donaldson, Begoña Diaz, Orit Gutman, G. Steven Martin, and Yoav I. Henis. "Src kinase activity and SH2 domain regulate the dynamics of Src association with lipid and protein targets." Journal of Cell Biology 178, no. 4 (August 13, 2007): 675–86. http://dx.doi.org/10.1083/jcb.200701133.
Full textAbstract:
Src functions depend on its association with the plasma membrane and with specific membrane-associated assemblies. Many aspects of these interactions are unclear. We investigated the functions of kinase, SH2, and SH3 domains in Src membrane interactions. We used FRAP beam-size analysis in live cells expressing a series of c-Src–GFP proteins with targeted mutations in specific domains together with biochemical experiments to determine whether the mutants can generate and bind to phosphotyrosyl proteins. Wild-type Src displays lipid-like membrane association, whereas constitutively active Src-Y527F interacts transiently with slower-diffusing membrane-associated proteins. These interactions require Src kinase activity and SH2 binding, but not SH3 binding. Furthermore, overexpression of paxillin, an Src substrate with a high cytoplasmic population, competes with membrane phosphotyrosyl protein targets for binding to activated Src. Our observations indicate that the interactions of Src with lipid and protein targets are dynamic and that the kinase and SH2 domain cooperate in the membrane targeting of Src.
31
Auvinen, M., A. Paasinen-Sohns, H. Hirai, L. C. Andersson, and E. Hölttä. "Ornithine decarboxylase- and ras-induced cell transformations: reversal by protein tyrosine kinase inhibitors and role of pp130CAS." Molecular and Cellular Biology 15, no. 12 (December 1995): 6513–25. http://dx.doi.org/10.1128/mcb.15.12.6513.
Full textAbstract:
We have found that overexpression of human ornithine decarboxylase (ODC) induces cell transformation in NIH 3T3 and Rat-1 cells (M. Auvinen, A. Paasinen, L. C. Andersson, and E. Hölttä, Nature (London) 360:355-358, 1992). The ODC-transformed cells display increased levels of tyrosine phosphorylation, in particular of a cluster of 130-kDa proteins. Here we show that one of the proteins with enhanced levels of tyrosine phosphorylation in ODC-overexpressing cells is the previously described p130 substrate of pp60v-src, known to associate also with v-Crk and designated p130CAS. We also studied the role of protein tyrosine phosphorylation in the ODC-induced cell transformation by exposing the cells to herbimycin A, a potent inhibitor of Src-family kinases, and to other inhibitors of protein tyrosine kinases. Treatment with the inhibitors reversed the phenotype of ODC-transformed cells to normal, with an organized, filamentous actin cytoskeleton. Coincidentally, the tyrosine hyperphosphorylation of p130 was markedly reduced, while the level of activity of ODC remained highly elevated. A similar reduction in pp130 phosphorylation and reversion of morphology by herbimycin A were observed in v-src- and c-Ha-ras-transformed cells. In addition, we show that expression of antisense mRNA for p130CAS resulted in reversion of the transformed phenotype of all these cell lines. An increased level of tyrosine kinase activity, not caused by c-Src or c-Abl, was further detected in the cytoplasmic fraction of ODC-transformed cells. Preliminary characteristics of this kinase are shown. These data indicate that p130CAS is involved in cell transformation by ODC, c-ras, and v-src oncogenes, raise the intriguing possibility that p130CAS may be generally required for transformation, and imply that there is at least one protein tyrosine kinase downstream of ODC that is instrumental for cell transformation.
32
Jha, Vibhu, Marco Macchia, Tiziano Tuccinardi, and Giulio Poli. "Three-Dimensional Interactions Analysis of the Anticancer Target c-Src Kinase with Its Inhibitors." Cancers 12, no. 8 (August 18, 2020): 2327. http://dx.doi.org/10.3390/cancers12082327.
Full textAbstract:
Src family kinases (SFKs) constitute the biggest family of non-receptor tyrosine kinases considered as therapeutic targets for cancer therapy. An aberrant expression and/or activation of the proto-oncogene c-Src kinase, which is the oldest and most studied member of the family, has long been demonstrated to play a major role in the development, growth, progression and metastasis of numerous human cancers, including colon, breast, gastric, pancreatic, lung and brain carcinomas. For these reasons, the pharmacological inhibition of c-Src activity represents an effective anticancer strategy and a few compounds targeting c-Src, together with other kinases, have been approved as drugs for cancer therapy, while others are currently undergoing preclinical studies. Nevertheless, the development of potent and selective inhibitors of c-Src aimed at properly exploiting this biological target for the treatment of cancer still represents a growing field of study. In this review, the co-crystal structures of c-Src kinase in complex with inhibitors discovered in the past two decades have been described, highlighting the key ligand–protein interactions necessary to obtain high potency and the features to be exploited for addressing selectivity and drug resistance issues, thus providing useful information for the design of new and potent c-Src kinase inhibitors.
33
Dobkin-Bekman, Masha, Michal Naidich, Liat Rahamim, Fiorenza Przedecki, Tal Almog, Stefan Lim, Philippa Melamed, et al. "A Preformed Signaling Complex Mediates GnRH-Activated ERK Phosphorylation of Paxillin and FAK at Focal Adhesions in LβT2 Gonadotrope Cells." Molecular Endocrinology 23, no. 11 (November 1, 2009): 1850–64. http://dx.doi.org/10.1210/me.2008-0260.
Full textAbstract:
Abstract Most receptor tyrosine kinases and G protein-coupled receptors (GPCRs) operate via a limited number of MAPK cascades but still exert diverse functions, and therefore signal specificity remains an enigma. Also, most GPCR ligands utilize families of receptors for mediation of diverse biological actions; however, the mammalian type I GnRH receptor (GnRHR) seems to be the sole receptor mediating GnRH-induced gonadotropin synthesis and release. Signaling complexes associated with GPCRs may thus provide the means for signal specificity. Here we describe a signaling complex associated with the GnRHR, which is a unique GPCR lacking a C-terminal tail. Unlike other GPCRs, this signaling complex is preformed, and exposure of LβT2 gonadotropes to GnRH induces its dynamic rearrangement. The signaling complex includes c-Src, protein kinase Cδ, -ε, and -α, Ras, MAPK kinase 1/2, ERK1/2, tubulin, focal adhesion kinase (FAK), paxillin, vinculin, caveolin-1, kinase suppressor of Ras-1, and the GnRHR. Exposure to GnRH (5 min) causes MAPK kinase 1/2, ERK1/2, tubulin, vinculin, and the GnRHR to detach from c-Src, but they reassociate within 30 min. On the other hand, FAK, paxillin, the protein kinase Cs, and caveolin-1 stay bound to c-Src, whereas kinase suppressor of Ras-1 appears in the complex only 30 min after GnRH stimulation. GnRH was found to activate ERK1/2 in the complex in a c-Src-dependent manner, and the activated ERK1/2 subsequently phosphorylates FAK and paxillin. In parallel, caveolin-1, FAK, vinculin, and paxillin are phosphorylated on Tyr residues apparently by GnRH-activated c-Src. Receptor tyrosine kinases and GPCRs translocate ERK1/2 to the nucleus to phosphorylate and activate transcription factors. We therefore propose that the role of the multiprotein signaling complex is to sequester a cytosolic pool of activated ERK1/2 to phosphorylate FAK and paxillin at focal adhesions.
34
Pleiman, C. M., M. R. Clark, L. K. Gauen, S. Winitz, K. M. Coggeshall, G. L. Johnson, A. S. Shaw, and J. C. Cambier. "Mapping of sites on the Src family protein tyrosine kinases p55blk, p59fyn, and p56lyn which interact with the effector molecules phospholipase C-gamma 2, microtubule-associated protein kinase, GTPase-activating protein, and phosphatidylinositol 3-kinase." Molecular and Cellular Biology 13, no. 9 (September 1993): 5877–87. http://dx.doi.org/10.1128/mcb.13.9.5877-5887.1993.
Full textAbstract:
Engagement of the B-cell antigen receptor complex induces immediate activation of receptor-associated Src family tyrosine kinases including p55blk, p59fyn, p53/56lyn, and perhaps p56lck, and this response is accompanied by tyrosine phosphorylation of distinct cellular substrates. These kinases act directly or indirectly to phosphorylate and/or activate effector proteins including p42 (microtubule-associated protein kinase) (MAPK), phospholipases C-gamma 1 (PLC gamma 1) and C-gamma 2 (PLC gamma 2), phosphatidylinositol 3-kinase (PI 3-K), and p21ras-GTPase-activating protein (GAP). Although coimmunoprecipitation results indicate that the Src family protein tyrosine kinases interact physically with some of these effector molecules, the molecular basis of this interaction has not been established. Here, we show that three distinct sites mediate the interaction of these kinases with effectors. The amino-terminal 27 residues of the unique domain of p56lyn mediate association with PLC gamma 2, MAPK, and GAP. Binding to PI 3-K is mediated through the Src homology 3 (SH3) domains of the Src family kinases. Relatively small proportions of cellular PI 3-K, PLC gamma 2, MAPK, and GAP, presumably those which are tyrosine phosphorylated, bind to the SH2 domains of these kinases. Comparative analysis of binding activities of Blk, Lyn, and Fyn shows that these kinases differ in their abilities to associate with MAPK and PI 3-K, suggesting that they may preferentially bind and subsequently phosphorylate distinct sets of downstream effector molecules in vivo. Fast protein liquid chromatography Mono Q column-fractionated MAPK maintains the ability to bind bacterially expressed Lyn, suggesting that the two kinases may interact directly.
35
Pleiman, C. M., M. R. Clark, L. K. Gauen, S. Winitz, K. M. Coggeshall, G. L. Johnson, A. S. Shaw, and J. C. Cambier. "Mapping of sites on the Src family protein tyrosine kinases p55blk, p59fyn, and p56lyn which interact with the effector molecules phospholipase C-gamma 2, microtubule-associated protein kinase, GTPase-activating protein, and phosphatidylinositol 3-kinase." Molecular and Cellular Biology 13, no. 9 (September 1993): 5877–87. http://dx.doi.org/10.1128/mcb.13.9.5877.
Full textAbstract:
Engagement of the B-cell antigen receptor complex induces immediate activation of receptor-associated Src family tyrosine kinases including p55blk, p59fyn, p53/56lyn, and perhaps p56lck, and this response is accompanied by tyrosine phosphorylation of distinct cellular substrates. These kinases act directly or indirectly to phosphorylate and/or activate effector proteins including p42 (microtubule-associated protein kinase) (MAPK), phospholipases C-gamma 1 (PLC gamma 1) and C-gamma 2 (PLC gamma 2), phosphatidylinositol 3-kinase (PI 3-K), and p21ras-GTPase-activating protein (GAP). Although coimmunoprecipitation results indicate that the Src family protein tyrosine kinases interact physically with some of these effector molecules, the molecular basis of this interaction has not been established. Here, we show that three distinct sites mediate the interaction of these kinases with effectors. The amino-terminal 27 residues of the unique domain of p56lyn mediate association with PLC gamma 2, MAPK, and GAP. Binding to PI 3-K is mediated through the Src homology 3 (SH3) domains of the Src family kinases. Relatively small proportions of cellular PI 3-K, PLC gamma 2, MAPK, and GAP, presumably those which are tyrosine phosphorylated, bind to the SH2 domains of these kinases. Comparative analysis of binding activities of Blk, Lyn, and Fyn shows that these kinases differ in their abilities to associate with MAPK and PI 3-K, suggesting that they may preferentially bind and subsequently phosphorylate distinct sets of downstream effector molecules in vivo. Fast protein liquid chromatography Mono Q column-fractionated MAPK maintains the ability to bind bacterially expressed Lyn, suggesting that the two kinases may interact directly.
36
Neet, K., and T. Hunter. "The nonreceptor protein-tyrosine kinase CSK complexes directly with the GTPase-activating protein-associated p62 protein in cells expressing v-Src or activated c-Src." Molecular and Cellular Biology 15, no. 9 (September 1995): 4908–20. http://dx.doi.org/10.1128/mcb.15.9.4908.
Full textAbstract:
CSK is a predominantly cytosolic protein-tyrosine kinase (PTK) that negatively regulates Src family PTKs by phosphorylation of a conserved tyrosine near their C termini. Little is known about how CSK itself is regulated. On the basis of immunofluorescence studies, a model has been proposed that when c-Src is activated, it is redistributed to podosomes, in which substrates become phosphorylated, creating binding sites for CSK. CSK is recruited to these sites of c-Src activation via its SH2 and SH3 domains and is then in a position to downregulate c-Src activity (B. W. Howell and J. A. Cooper, Mol. Cell. Biol. 14:5402-5411, 1994). To identify phosphotyrosine (P.Tyr)-containing proteins that may mediate translocation of CSK due to c-Src activation, we have examined the whole spectrum of P.Tyr-containing proteins that associate with CSK in v-Src NIH 3T3 cells by anti-P.Tyr immunoblotting. Nine P.Tyr-containing proteins coimmunoprecipitated with CSK from v-Src NIH 3T3 cells. One of these, an approximately 62-kDa protein, also associated with CSK in NIH 3T3 cells treated with vanadate prior to lysis and in NIH 3T3 cells expressing an activated c-Src mutant. This 62-kDa protein was shown to be identical to the GTPase-activating protein (GAP)-associated p62 (GAP-A.p62) protein. The interaction between CSK and GAP-A.p62 could be reconstituted in vitro with glutathione S-transferase fusion proteins containing full-length CSK or the CSK SH2 domain. Furthermore, our data show that CSK interacts directly with GAP.A-p62 and that the complex between the two proteins is localized in subcellular membrane or cytoskeletal fractions. Our results suggest that GAP-A.p62 may function as a docking protein and may mediate translocation of proteins, including GAP and CSK, to membrane or cytoskeletal regions upon c-Src activation.
37
Fujii, M., D. Shalloway, and I. M. Verma. "Gene regulation by tyrosine kinases: src protein activates various promoters, including c-fos." Molecular and Cellular Biology 9, no. 6 (June 1989): 2493–99. http://dx.doi.org/10.1128/mcb.9.6.2493-2499.1989.
Full textAbstract:
A promoter of the nuclear proto-oncogene fos was activated by cotransfection with the viral src gene. Ability to transactivate the c-fos promoter was dependent on tyrosine kinase activity, because (i) src mutants which have reduced tyrosine kinase activity due to mutation of Tyr-416 to Phe showed lower promoter activation, (ii) pp60c-src mutants which have increased tyrosine kinase activity due to mutation of Tyr-527 to Phe also augmented c-fos promoter induction, and (iii) mutation in the ATP-binding site of pp60v-src strongly suppressed c-fos promoter activation. Tyrosine kinase activity alone, however, was not sufficient for promoter activation, because of pp60v-src mutant which lacked its myristylation site and consequently membrane association showed no increased c-fos promoter activation. Both the tyrosine kinase- and membrane-association-defective mutants were also unable to induce transformation. Therefore, phosphorylation of membrane-associated substrates appears to be required for both gene expression and cellular transformation by the src protein. Two regions of the c-fos promoter located between positions -362 and -324 and positions -323 and -294 were responsive to src stimulation. We believe that protein tyrosine phosphorylation represents an important step of signal transduction from the membrane to the nucleus.
38
Fujii, M., D. Shalloway, and I. M. Verma. "Gene regulation by tyrosine kinases: src protein activates various promoters, including c-fos." Molecular and Cellular Biology 9, no. 6 (June 1989): 2493–99. http://dx.doi.org/10.1128/mcb.9.6.2493.
Full textAbstract:
A promoter of the nuclear proto-oncogene fos was activated by cotransfection with the viral src gene. Ability to transactivate the c-fos promoter was dependent on tyrosine kinase activity, because (i) src mutants which have reduced tyrosine kinase activity due to mutation of Tyr-416 to Phe showed lower promoter activation, (ii) pp60c-src mutants which have increased tyrosine kinase activity due to mutation of Tyr-527 to Phe also augmented c-fos promoter induction, and (iii) mutation in the ATP-binding site of pp60v-src strongly suppressed c-fos promoter activation. Tyrosine kinase activity alone, however, was not sufficient for promoter activation, because of pp60v-src mutant which lacked its myristylation site and consequently membrane association showed no increased c-fos promoter activation. Both the tyrosine kinase- and membrane-association-defective mutants were also unable to induce transformation. Therefore, phosphorylation of membrane-associated substrates appears to be required for both gene expression and cellular transformation by the src protein. Two regions of the c-fos promoter located between positions -362 and -324 and positions -323 and -294 were responsive to src stimulation. We believe that protein tyrosine phosphorylation represents an important step of signal transduction from the membrane to the nucleus.
39
Wang, H. C., and R. L. Erikson. "Activation of protein serine/threonine kinases p42, p63, and p87 in Rous sarcoma virus-transformed cells: signal transduction/transformation-dependent MBP kinases." Molecular Biology of the Cell 3, no. 12 (December 1992): 1329–37. http://dx.doi.org/10.1091/mbc.3.12.1329.
Full textAbstract:
We have used myelin basic protein immobilized in sodium dodecyl sulfate-polyacrylamide gels to identify protein kinases after gel electrophoresis, followed by protein kinase reactions. This technique has permitted us to detect three protein kinases in serum-deprived cells transformed by p60src. On induction of cellular transformation by a temperature-sensitive v-src, a p87 protein kinase is activated within 30 min and remains activated in fully transformed cells. The p63 protein kinase is not fully activated until 24 h but remains activated in transformed cells. The commonly studied p42MBPK is rapidly activated within 30 min, and its kinase activity decreases significantly by 24 h, when the p63 enzyme is fully activated. The p42MBPK, as well as the p63 and p87 enzymes, are stimulated by transforming p60c-src mutants but not normal c-src or nonmyristylated p60c-src. In addition, the kinase activity of p63 enzyme, but not of p42MBPK, can be induced in okadaic acid-treated chicken embryo fibroblasts, indicating that phosphatase 2A and/or phosphatase 1 may be involved in the regulation of its activity. Additional data indicate that either p42MBPK or p63 activity correlates with the stimulation of the protein kinase p90RSK. Thus, there may be two independent pathways leading to the activation of the RSK gene product.
40
Klein, N. P., and R. J. Schneider. "Activation of Src family kinases by hepatitis B virus HBx protein and coupled signaling to Ras." Molecular and Cellular Biology 17, no. 11 (November 1997): 6427–36. http://dx.doi.org/10.1128/mcb.17.11.6427.
Full textAbstract:
The HBx protein of hepatitis B virus (HBV) is a small transcriptional transactivator that is essential for infection by the mammalian hepadnaviruses and is thought to be a cofactor in HBV-mediated liver cancer. HBx stimulates signal transduction pathways by acting in the cytoplasm, which accounts for many but not all of its transcriptional activities. Studies have shown that HBx protein activates Ras and downstream Ras signaling pathways including Raf, mitogen-activated protein (MAP) kinase kinase kinase (MEK), and MAP kinases. In this study, we investigated the mechanism of activation of Ras by HBx because it has been found to be central to the ability of HBx protein to stimulate transcription and to release growth arrest in quiescent cells. In contrast to the transient but strong stimulation of Ras typical of autocrine factors, activation of Ras by HBx protein was found to be constitutive but moderate. HBx induced the association of Ras upstream activating proteins Shc, Grb2, and Sos and stimulated GTP loading onto Ras, but without directly participating in complex formation. Instead, HBx is shown to stimulate Ras-activating proteins by functioning as an intracellular cytoplasmic activator of the Src family of tyrosine kinases, which can signal to Ras. HBx protein stimulated c-Src and Fyn kinases for a prolonged time. Activation of Src is shown to be indispensable for a number of HBx activities, including activation of Ras and the Ras-Raf-MAP kinase pathway and stimulation of transcription mediated by transcription factor AP-1. Importantly, HBx protein expressed in cultured cells during HBV replication is shown to activate the Ras signaling pathway. Mechanisms by which HBx protein might activate Src kinases are discussed.
41
Bell, S. M., D. C. Connolly, N. J. Maihle, and J. L. Degen. "Differential modulation of plasminogen activator gene expression by oncogene-encoded protein tyrosine kinases." Molecular and Cellular Biology 13, no. 9 (September 1993): 5888–97. http://dx.doi.org/10.1128/mcb.13.9.5888-5897.1993.
Full textAbstract:
Urokinase-type plasminogen activator (uPA) gene transcription is increased > or = 50-fold in chicken embryo fibroblasts (CEF) following transformation by the protein tyrosine kinase pp60v-src. Protein phosphorylation appears to play a critical role in uPA gene expression in these cells; protein kinase C-activating phorbol esters cooperate with pp60v-src to synergistically increase uPA mRNA, whereas cyclic AMP (cAMP)-dependent protein kinase-activating agents (e.g., 8-bromo cAMP) repress uPA mRNA levels. To explore the relationship between transforming oncogenes and uPA gene expression, uPA mRNA levels were measured in CEF infected with selected avian retroviruses. We report that v-ras and the transforming protein tyrosine kinases v-src, v-yes, and v-ros all increase cellular uPA mRNAs. However, transformation with the protein tyrosine kinase encoded by v-erbB, or the nuclear proteins encoded by v-jun, v-ski, or v-myc, did not increase uPA mRNA detectably. Ras and all of the protein tyrosine kinases analyzed, including the v-erbB product, but none of the nuclear oncoproteins sensitized cells to phorbol ester induction of uPA gene expression. Thus, increased uPA gene expression is not simply a secondary consequence of cell transformation but, rather, is regulated or comodulated by only a subset of oncogene products. Analysis of cells expressing site-directed mutants of pp60v-src showed that the induction of the uPA gene is dependent on protein tyrosine kinase catalytic activity, myristylation, and plasma membrane localization. However, these properties together are not sufficient; an additional feature in the src homology 2 domain is also required. The major sites of serine phosphorylation, serines 12 and 17, and the autophosphorylation site, tyrosine 416, are not essential for uPA gene induction. However, the reduction of uPA mRNA in pp60v-src-transformed cells by 8-bromo cAMP is dependent on tyrosine 416.
42
Bell, S. M., D. C. Connolly, N. J. Maihle, and J. L. Degen. "Differential modulation of plasminogen activator gene expression by oncogene-encoded protein tyrosine kinases." Molecular and Cellular Biology 13, no. 9 (September 1993): 5888–97. http://dx.doi.org/10.1128/mcb.13.9.5888.
Full textAbstract:
Urokinase-type plasminogen activator (uPA) gene transcription is increased > or = 50-fold in chicken embryo fibroblasts (CEF) following transformation by the protein tyrosine kinase pp60v-src. Protein phosphorylation appears to play a critical role in uPA gene expression in these cells; protein kinase C-activating phorbol esters cooperate with pp60v-src to synergistically increase uPA mRNA, whereas cyclic AMP (cAMP)-dependent protein kinase-activating agents (e.g., 8-bromo cAMP) repress uPA mRNA levels. To explore the relationship between transforming oncogenes and uPA gene expression, uPA mRNA levels were measured in CEF infected with selected avian retroviruses. We report that v-ras and the transforming protein tyrosine kinases v-src, v-yes, and v-ros all increase cellular uPA mRNAs. However, transformation with the protein tyrosine kinase encoded by v-erbB, or the nuclear proteins encoded by v-jun, v-ski, or v-myc, did not increase uPA mRNA detectably. Ras and all of the protein tyrosine kinases analyzed, including the v-erbB product, but none of the nuclear oncoproteins sensitized cells to phorbol ester induction of uPA gene expression. Thus, increased uPA gene expression is not simply a secondary consequence of cell transformation but, rather, is regulated or comodulated by only a subset of oncogene products. Analysis of cells expressing site-directed mutants of pp60v-src showed that the induction of the uPA gene is dependent on protein tyrosine kinase catalytic activity, myristylation, and plasma membrane localization. However, these properties together are not sufficient; an additional feature in the src homology 2 domain is also required. The major sites of serine phosphorylation, serines 12 and 17, and the autophosphorylation site, tyrosine 416, are not essential for uPA gene induction. However, the reduction of uPA mRNA in pp60v-src-transformed cells by 8-bromo cAMP is dependent on tyrosine 416.
43
Yamaji, Y., H. Tsuganezawa, O. W. Moe, and R. J. Alpern. "Intracellular acidosis activates c-Src." American Journal of Physiology-Cell Physiology 272, no. 3 (March 1, 1997): C886—C893. http://dx.doi.org/10.1152/ajpcell.1997.272.3.c886.
Full textAbstract:
The purpose of the present studies was to determine whether acidosis activates protein tyrosine kinase pathways. Incubation of MCT cells, a renal proximal tubule cell line, in acid media caused increased phosphotyrosine content of 60- to 70- and 120-kDa cytosolic proteins. Media acidification induced a twofold increase in c-Src activity that occurred within 30 s. Significant activation occurred with media pH changes as small as 0.07 pH unit accompanied by cell acidification of 0.06 pH unit. Sodium propionate addition, NH4Cl prepulse, and nigericin addition, maneuvers that decrease intracellular pH in the absence of changes in extracellular pH, activated c-Src. Significant activation by sodium propionate was seen with cell pH changes as small as 0.07 pH unit. Sodium orthovanadate, a protein tyrosine phosphatase inhibitor, prevented c-Src activation by media acidification but did not prevent protein tyrosine phosphorylation. In summary, decreased intracellular pH activates c-Src. Acid activation of c-Src represents a novel mechanism of c-Src activation that may be relevant to many cellular responses to acidosis.
44
Pertel, Thomas, Defen Zhu, Reynold A. Panettieri, Naoto Yamaguchi, Charles W. Emala, and Carol A. Hirshman. "Expression and muscarinic receptor coupling of Lyn kinase in cultured human airway smooth muscle cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 290, no. 3 (March 2006): L492—L500. http://dx.doi.org/10.1152/ajplung.00344.2005.
Full textAbstract:
Src family tyrosine kinases are signaling intermediates in a diverse array of cellular events including cell differentiation, motility, proliferation, and survival. In nonairway smooth muscle cells, muscarinic receptors directly interact with Src family tyrosine kinases. As little is known about the expression and signaling of these Src family tyrosine kinases in human airway smooth muscle cells, we determined the expression of Src family members and characterized the muscarinic receptor-mediated activation of Lyn kinase in these cells. RT-PCR revealed mRNA transcripts for FYN, c- SRC, YES, FRK, and LYN. Fyn, c-Src, Yes, and Lyn were identified in cultured airway smooth muscle cells by immunoblot analysis. In both nontransformed human cultured airway smooth muscle cells and cells transduced with wild-type human Lyn kinase, carbachol increased Lyn kinase activity. Pertussis toxin pretreatment failed to block carbachol activation of Lyn kinase but did attenuate the carbachol-induced increase in ERK/MAPK phosphorylation. Moreover, carbachol inhibited adenylyl cyclase but failed to increase total inositol phosphate synthesis in these cells. The present study shows that Lyn kinase is expressed in human cultured airway smooth muscle cells at both the mRNA and protein levels and that carbachol, an M2 muscarinic receptor agonist in these cells, activates Lyn kinase by a pertussis toxin-insensitive signaling pathway.
45
Gatesman, Amanda, Valerie G. Walker, Joseph M. Baisden, Scott A. Weed, and Daniel C. Flynn. "Protein Kinase Cα Activates c-Src and Induces Podosome Formation via AFAP-110." Molecular and Cellular Biology 24, no. 17 (September 1, 2004): 7578–97. http://dx.doi.org/10.1128/mcb.24.17.7578-7597.2004.
Full textAbstract:
ABSTRACT We report that the actin filament-associated protein AFAP-110 is required to mediate protein kinase Cα (PKCα) activation of the nonreceptor tyrosine kinase c-Src and the subsequent formation of podosomes. Immunofluorescence analysis demonstrated that activation of PKCα by phorbol 12-myristate 13-acetate (PMA), or ectopic expression of constitutively activated PKCα, directs AFAP-110 to colocalize with and bind to the c-Src SH3 domain, resulting in activation of the tyrosine kinase. Activation of c-Src then directs the formation of podosomes, which contain cortactin, AFAP-110, actin, and c-Src. In a cell line (CaOV3) that has very little or no detectable AFAP-110, PMA treatment was unable to activate c-Src or effect podosome formation. Ectopic expression of AFAP-110 in CaOV3 cells rescued PKCα-mediated activation of c-Src and elevated tyrosine phosphorylation levels and subsequent formation of podosomes. Neither expression of activated PKCα nor treatment with PMA was able to induce these changes in CAOV3 cells expressing mutant forms of AFAP-110 that are unable to bind to, or colocalize with, c-Src. We hypothesize that one major function of AFAP-110 is to relay signals from PKCα that direct the activation of c-Src and the formation of podosomes.
46
Liebenhoff, U., D. Brockmeier, and P. Presek. "Substrate affinity of the protein tyrosine kinase pp60c-src is increased on thrombin stimulation of human platelets." Biochemical Journal 295, no. 1 (October 1, 1993): 41–48. http://dx.doi.org/10.1042/bj2950041.
Full textAbstract:
Human blood platelets contain high levels of non-receptor protein tyrosine kinases of the Src family, particularly pp60c-src, suggesting an important role for these enzymes in platelet physiology. Indeed, in response to various agonists of platelet function, a number of proteins become phosphorylated at tyrosine residues. However, no enzymic activation of an Src-related tyrosine kinase has yet been shown in platelets. In searching for the kinase(s) responsible, we found that all agonists tested that directly or indirectly activate protein kinase C in platelets (phorbol 12-myristate, 13-acetate, thrombin, vasopressin, collagen, calcium ionophore A23187) increased the overall activity of pp60c-src determined by IgG phosphorylation in an immunocomplex assay in the presence of low ATP concentrations. On the other hand, elevation of cyclic AMP directly by forskolin or indirectly by prostaglandin E1, or elevation of cyclic GMP by sodium nitroprusside did not significantly affect the activity of the enzyme. To substantiate the differences in enzyme activity, we determined Km and Vmax, values of pp60c-src from resting and thrombin-stimulated platelets. Thrombin treatment increased substrate affinity of pp60c-src as indicated by a 2- to 3-fold decrease in the Km values for ATP and the exogenous protein substrate casein. Vmax. values were only slightly altered under the assay conditions used. To further rule out modifications of pp60c-src in phosphorylation as a probable cause of the changed substrate affinity, we analysed tryptic phosphopeptides of immunoprecipitated, 32P-labelled pp60c-src of unstimulated and stimulated platelets. The platelet agonists listed above induced an increase in pp60c-src phosphorylation at Ser-12, which is the amino acid phosphorylated by protein kinase C. Surprisingly, we found that elevation of cyclic AMP did not affect 32P labelling of pp60c-src. On the basis of our data, we suggest that phosphorylation at Ser-12 might be one of the signal-triggering events that cause the increase in substrate affinity of pp60c-src.
47
Dong, Su, Andrew Khoo, Jianxin Wei, Rachel K. Bowser, Nathaniel M. Weathington, Shuqi Xiao, Lina Zhang, Haichun Ma, Yutong Zhao, and Jing Zhao. "Serum starvation regulates E-cadherin upregulation via activation of c-Src in non-small-cell lung cancer A549 cells." American Journal of Physiology-Cell Physiology 307, no. 9 (November 1, 2014): C893—C899. http://dx.doi.org/10.1152/ajpcell.00132.2014.
Full textAbstract:
E-cadherin is essential for the integrity of adherens junctions between lung epithelial cells, and the loss of E-cadherin allows cell motility and is thought to promote lung cancer metastasis. While the downregulation of E-cadherin expression has been well characterized and is seen with transforming growth factor-β1 (TGF-β1) exposure, few studies have focused on E-cadherin upregulation. Here, we show that serum starvation causes increased E-cadherin expression via the activation of c-Src kinase in non-small-cell lung cancer A549 cells. Serum starvation increased E-cadherin protein levels in a time- and dose-dependent manner. E-cadherin mRNA transcripts were unchanged with starvation, while protein translation inhibition with cycloheximide attenuated E-cadherin protein induction by starvation, suggesting that E-cadherin is regulated at the translational level by serum starvation. c-Src is a nonreceptor tyrosine kinase known to regulate protein translation machinery; serum starvation caused early and sustained activation of c-Src in A549 cells followed by E-cadherin upregulation. Furthermore, overexpression of a dominant negative c-Src attenuated the induction of E-cadherin by serum deprivation. Finally, we observed that TGF-β1 treatment attenuated the serum activation of c-Src as well as E-cadherin expression when cells were deprived of serum. In conclusion, our data demonstrate that the c-Src kinase is activated by serum starvation to increase E-cadherin expression in A549 cells, and these phenomena are antagonized by TGF-β1. These novel observations implicate the c-Src kinase as an upstream inducer of E-cadherin protein translation with serum starvation and TGF-β1 diametrically regulating c-Src kinase activity and thus E-cadherin abundance in A549 cells.
48
Seidel-Dugan, C., B. E. Meyer, S. M. Thomas, and J. S. Brugge. "Effects of SH2 and SH3 deletions on the functional activities of wild-type and transforming variants of c-Src." Molecular and Cellular Biology 12, no. 4 (April 1992): 1835–45. http://dx.doi.org/10.1128/mcb.12.4.1835-1845.1992.
Full textAbstract:
The amino-termina, noncatalytic half of Src contains two domains, designated the Src homology 2 (SH2) and Src homology 3 (SH3) domains, that are highly conserved among members of the Src family of tyrosine kinases. The SH2 domain (which can be further divided into the B and C homology boxes) and the SH3 domain (also referred to as the A box) are also found in several proteins otherwise unrelated to protein tyrosine kinases. It is believed that these domains are important for directing specific protein-protein interactions necessary for the proper functioning of Src. To determine the importance of the SH2 and SH3 domains in regulating the functions of c-Src, we evaluated mutants of c-Src lacking the A box (residues 88 to 137), the B box (residues 148 to 187) or the C box (residues 220 to 231). Each of these deletions caused a 14- to 30-fold increase in the in vitro level of kinase activity of c-Src. Chicken embryo fibroblasts expressing the deletion mutants displayed a transformed cell morphology, formed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Src substrates p36, p85, p120, p125, the GTPase-activating protein (GAP), and several GAP-associated proteins were phosphorylated on tyrosine in cells expressing the A, B, or C box deletion mutant. p110 was highly phosphorylated in cells expressing the C box mutant, was weakly phosphorylated in cells expressing the B box mutant, and was not phosphorylated in cells expressing the A box mutant. Expression of the mutant proteins caused a reorganization of the actin cytoskeleton similar to that seen in v-Src-transformed cells. In addition, deletion of the A, B, or C box did not diminish the transforming or enzymatic activity of an activated variant of c-Src, E378G. These data indicate that deletion of the A, B, or C homology box causes an activation of the catalytic and transforming potential of c-Src and that while these mutations caused subtle differences in substrate phosphorylation, the homology boxes are not required for many of the phenotypic changes associated with transformation by Src.
49
Seidel-Dugan, C., B. E. Meyer, S. M. Thomas, and J. S. Brugge. "Effects of SH2 and SH3 deletions on the functional activities of wild-type and transforming variants of c-Src." Molecular and Cellular Biology 12, no. 4 (April 1992): 1835–45. http://dx.doi.org/10.1128/mcb.12.4.1835.
Full textAbstract:
The amino-termina, noncatalytic half of Src contains two domains, designated the Src homology 2 (SH2) and Src homology 3 (SH3) domains, that are highly conserved among members of the Src family of tyrosine kinases. The SH2 domain (which can be further divided into the B and C homology boxes) and the SH3 domain (also referred to as the A box) are also found in several proteins otherwise unrelated to protein tyrosine kinases. It is believed that these domains are important for directing specific protein-protein interactions necessary for the proper functioning of Src. To determine the importance of the SH2 and SH3 domains in regulating the functions of c-Src, we evaluated mutants of c-Src lacking the A box (residues 88 to 137), the B box (residues 148 to 187) or the C box (residues 220 to 231). Each of these deletions caused a 14- to 30-fold increase in the in vitro level of kinase activity of c-Src. Chicken embryo fibroblasts expressing the deletion mutants displayed a transformed cell morphology, formed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Src substrates p36, p85, p120, p125, the GTPase-activating protein (GAP), and several GAP-associated proteins were phosphorylated on tyrosine in cells expressing the A, B, or C box deletion mutant. p110 was highly phosphorylated in cells expressing the C box mutant, was weakly phosphorylated in cells expressing the B box mutant, and was not phosphorylated in cells expressing the A box mutant. Expression of the mutant proteins caused a reorganization of the actin cytoskeleton similar to that seen in v-Src-transformed cells. In addition, deletion of the A, B, or C box did not diminish the transforming or enzymatic activity of an activated variant of c-Src, E378G. These data indicate that deletion of the A, B, or C homology box causes an activation of the catalytic and transforming potential of c-Src and that while these mutations caused subtle differences in substrate phosphorylation, the homology boxes are not required for many of the phenotypic changes associated with transformation by Src.
50
Mellström, K., C. Bjelfman, U. Hammerling, and S. Påhlman. "Expression of c-src in cultured human neuroblastoma and small-cell lung carcinoma cell lines correlates with neurocrine differentiation." Molecular and Cellular Biology 7, no. 12 (December 1987): 4178–84. http://dx.doi.org/10.1128/mcb.7.12.4178-4184.1987.
Full textAbstract:
Human cell lines with neuronal and neuroendocrine features were examined for their expression of pp60c-src, the cellular homolog of the transforming gene product pp60v-src of Rous sarcoma virus. Four neuroblastoma (LA-N-5, SH-SY5Y, Paju, and SK-N-MC) and three small-cell lung carcinoma (U-2020, U-1690, and U-1285) cell lines were selected on the basis of their stage of neurocrine differentiation, as determined by the expression of neuron-specific enolase. In an immune complex protein kinase assay, all seven cell lines displayed c-src kinase activity which was considerably higher than that found in nonneurocrine cells (human diploid fibroblasts, glioma, and non-small cell lung carcinoma cell lines). Furthermore, the c-src kinase activity, as determined by autophosphorylation or phosphorylation of an exogenous substrate, enolase, correlated with the stage of neurocrine differentiation. There was an approximately 30-fold difference in c-src kinase autophosphorylation activity between the cell lines representing the highest and lowest stages of neurocrine differentiation. A similar variation was found in the steady-state levels of the c-src protein of these cell lines. Highly differentiated neuroblastoma cells expressed two forms of the src protein. Digestion by Staphylococcus aureus V8 protease did reveal structural diversity in the amino-terminal ends of these c-src molecules. In summary, we found a clear correlation between c-src kinase activity and the stage of neuronal and neuroendocrine differentiation. Thus, the phenotypic similarity between neurons and neuroendocrine cells includes high c-src expression.