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1
Clarke, Dominic M., Michael C. Brown, David P. LaLonde, and Christopher E. Turner. "Phosphorylation of actopaxin regulates cell spreading and migration." Journal of Cell Biology 166, no. 6 (September 7, 2004): 901–12. http://dx.doi.org/10.1083/jcb.200404024.
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Actopaxin is an actin and paxillin binding protein that localizes to focal adhesions. It regulates cell spreading and is phosphorylated during mitosis. Herein, we identify a role for actopaxin phosphorylation in cell spreading and migration. Stable clones of U2OS cells expressing actopaxin wild-type (WT), nonphosphorylatable, and phosphomimetic mutants were developed to evaluate actopaxin function. All proteins targeted to focal adhesions, however the nonphosphorylatable mutant inhibited spreading whereas the phosphomimetic mutant cells spread more efficiently than WT cells. Endogenous and WT actopaxin, but not the nonphosphorylatable mutant, were phosphorylated in vivo during cell adhesion/spreading. Expression of the nonphosphorylatable actopaxin mutant significantly reduced cell migration, whereas expression of the phosphomimetic increased cell migration in scrape wound and Boyden chamber migration assays. In vitro kinase assays demonstrate that extracellular signal-regulated protein kinase phosphorylates actopaxin, and treatment of U2OS cells with the MEK1 inhibitor UO126 inhibited adhesion-induced phosphorylation of actopaxin and also inhibited cell migration.
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Woodman, Julie, Matthew Hoffman, Monika Dzieciatkowska, Kirk C. Hansen, and Paul C. Megee. "Phosphorylation of the Scc2 cohesin deposition complex subunit regulates chromosome condensation through cohesin integrity." Molecular Biology of the Cell 26, no. 21 (November 2015): 3754–67. http://dx.doi.org/10.1091/mbc.e15-03-0165.
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The cohesion of replicated sister chromatids promotes chromosome biorientation, gene regulation, DNA repair, and chromosome condensation. Cohesion is mediated by cohesin, which is deposited on chromosomes by a separate conserved loading complex composed of Scc2 and Scc4 in Saccharomyces cerevisiae. Although it is known to be required, the role of Scc2/Scc4 in cohesin deposition remains enigmatic. Scc2 is a phosphoprotein, although the functions of phosphorylation in deposition are unknown. We identified 11 phosphorylated residues in Scc2 by mass spectrometry. Mutants of SCC2 with substitutions that mimic constitutive phosphorylation retain normal Scc2–Scc4 interactions and chromatin association but exhibit decreased viability, sensitivity to genotoxic agents, and decreased stability of the Mcd1 cohesin subunit in mitotic cells. Cohesin association on chromosome arms, but not pericentromeric regions, is reduced in the phosphomimetic mutants but remains above a key threshold, as cohesion is only modestly perturbed. However, these scc2 phosphomimetic mutants exhibit dramatic chromosome condensation defects that are likely responsible for their high inviability. From these data, we conclude that normal Scc2 function requires modulation of its phosphorylation state and suggest that scc2 phosphomimetic mutants cause an increased incidence of abortive cohesin deposition events that result in compromised cohesin complex integrity and Mcd1 turnover.
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Rolli-Derkinderen, Malvyne, Gilles Toumaniantz, Pierre Pacaud, and Gervaise Loirand. "RhoA Phosphorylation Induces Rac1 Release from Guanine Dissociation Inhibitor α and Stimulation of Vascular Smooth Muscle Cell Migration." Molecular and Cellular Biology 30, no. 20 (August 9, 2010): 4786–96. http://dx.doi.org/10.1128/mcb.00381-10.
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ABSTRACT Although overactivation of RhoA is recognized as a common component of vascular disorders, the molecular mechanisms regulating RhoA activity in vascular smooth muscle cells (VSMC) are still unclear. We have previously shown that in VSMC, RhoA is phosphorylated on Ser188 by nitric oxide (NO)-stimulated cGMP-dependent kinase (PKG), which leads to RhoA-Rho kinase pathway inhibition. In this study, we showed that expression of phosphoresistant RhoA mutants prevented the stimulation of VSMC migration and adhesion induced by NO-PKG pathway activation. In contrast, under basal conditions, phosphomimetic RhoA mutants stimulated VSMC adhesion and migration through a signaling pathway requiring Rac1 and the Rho exchange factor Vav3. RhoA phosphorylation or phosphomimetic RhoA mutants induced Rac1 activation but did not activate Vav3. Indeed, phosphorylated RhoA or phosphomimetic mutants trapped guanine dissociation inhibitor α (GDIα), leading to the release of Rac1 and its translocation to the membrane, where it was then activated by the basal Vav3 nucleotide exchange activity. In vivo, RhoA phosphorylation induced by PKG activation in the aortas of rats treated with sildenafil induced dissociation of Rac1 from GDIα and activation of the Rac1 signaling pathway. These results suggest that the phosphorylation of RhoA represents a novel potent and physiological GDIα displacement factor that leads to Rac1 activation and regulation of Rac1-dependent VSMC functions.
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Du, Wei, Yun Zhou, Suzette Pike, and Qishen Pang. "Cdk1-Dependent Phosphorylation ofNPM Overrides G2/M Checkpoint and Increases Leukemic Blasts in Mice." Blood 112, no. 11 (November 16, 2008): 1322. http://dx.doi.org/10.1182/blood.v112.11.1322.1322.
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Abstract An elevated level of nucleophosmin (NPM) is often found in actively proliferative cells including human tumors. To identify the regulatory role for NPM phosphorylation in proliferation and cell cycle control, a series of mutants targeting the consensus cyclin-dependent kinase (CKD) phosphorylation sites was created to mimic or abrogate either single-site or multi-site phosphorylation. Cells expressing the phosphomimetic NPM mutants showed enhanced proliferation and G2/M cell-cycle transition; whereas nonphosphorylatable mutants induced G2/M cell-cycle arrest. Simultaneous inactivation of two CKD phosphorylation sites at Ser10 and Ser70 (S10A/S70A, NPM-AA) induced phosphorylation of Cdk1 at Tyr15 (Cdc2Tyr15) and increased cytoplasmic accumulation of Cdc25C. Strikingly, stress-induced Cdk1Tyr15 and Cdc25C sequestration were completely suppressed by expression of a double phosphomimetic NPM mutant (S10E/S70E, NPM-EE). Further analysis revealed that phosphorylation of NPM at both Ser10 and Ser70 sites were required for proper interaction between Cdk1 and Cdc25C in mitotic cells. Moreover, the NPM-EE mutant directly bound to Cdc25C and prevented phosphorylation of Cdc25C at Ser216 during mitosis. Finally, NPM-EE overrided stress-induced G2/M arrest, increased peripheral-blood blasts and splenomegaly in a NOD/SCID xenograft model, and promoted leukemia development in Fanconi mouse hematopoietic stem/progenitor cells. Thus, these findings reveal a novel function of NPM on regulation of cell-cycle progression, in which Cdk1-dependent phosphorylation of NPM controls cell-cycle progression at G2/M transition through modulation of Cdc25C activity.
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Callaci, Sandhya, Kylee Morrison, Xiangqiang Shao, Amber L. Schuh, Yueju Wang, John R. Yates, Jeff Hardin, and Anjon Audhya. "Phosphoregulation of the C. elegans cadherin–catenin complex." Biochemical Journal 472, no. 3 (November 27, 2015): 339–52. http://dx.doi.org/10.1042/bj20150410.
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Mass spectrometry analysis reveals that the three essential subunits of the cadherin–catenin complex are phosphorylated in vivo. Examination of phosphomimetic mutants in vitro suggests that phosphoregulation plays a key role in the assembly and disassembly of adherens junctions.
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Levy, Robin, Emily Gregory, Wade Borcherds, and Gary Daughdrill. "p53 Phosphomimetics Preserve Transient Secondary Structure but Reduce Binding to Mdm2 and MdmX." Biomolecules 9, no. 3 (March 2, 2019): 83. http://dx.doi.org/10.3390/biom9030083.
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The disordered p53 transactivation domain (p53TAD) contains specific levels of transient helical secondary structure that are necessary for its binding to the negative regulators, mouse double minute 2 (Mdm2) and MdmX. The interactions of p53 with Mdm2 and MdmX are also modulated by posttranslational modifications (PTMs) of p53TAD including phosphorylation at S15, T18 and S20 that inhibits p53-Mdm2 binding. It is unclear whether the levels of transient secondary structure in p53TAD are changed by phosphorylation or other PTMs. We used phosphomimetic mutants to determine if adding a negative charge at positions 15 and 18 has any effect on the transient secondary structure of p53TAD and protein-protein binding. Using a combination of biophysical and structural methods, we investigated the effects of single and multisite phosphomimetics on the transient secondary structure of p53TAD and its interaction with Mdm2, MdmX, and the KIX domain. The phosphomimetics reduced Mdm2 and MdmX binding affinity by 3–5-fold, but resulted in minimal changes in transient secondary structure, suggesting that the destabilizing effect of phosphorylation on the p53TAD-Mdm2 interaction is primarily electrostatic. Phosphomimetics had no effect on the p53-KIX interaction, suggesting that increased binding of phosphorylated p53 to KIX may be influenced by decreased competition with its negative regulators.
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Brand, Sue Ellen, Martha Scharlau, Lois Geren, Marissa Hendrix, Clayre Parson, Tyler Elmendorf, Earl Neel, et al. "Accelerated Evolution of Cytochrome c in Higher Primates, and Regulation of the Reaction between Cytochrome c and Cytochrome Oxidase by Phosphorylation." Cells 11, no. 24 (December 12, 2022): 4014. http://dx.doi.org/10.3390/cells11244014.
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Cytochrome c (Cc) underwent accelerated evolution from the stem of the anthropoid primates to humans. Of the 11 amino acid changes that occurred from horse Cc to human Cc, five were at Cc residues near the binding site of the Cc:CcO complex. Single-point mutants of horse and human Cc were made at each of these positions. The Cc:CcO dissociation constant KD of the horse mutants decreased in the order: T89E > native horse Cc > V11I Cc > Q12M > D50A > A83V > native human. The largest effect was observed for the mutants at residue 50, where the horse Cc D50A mutant decreased KD from 28.4 to 11.8 μM, and the human Cc A50D increased KD from 4.7 to 15.7 μM. To investigate the role of Cc phosphorylation in regulating the reaction with CcO, phosphomimetic human Cc mutants were prepared. The Cc T28E, S47E, and Y48E mutants increased the dissociation rate constant kd, decreased the formation rate constant kf, and increased the equilibrium dissociation constant KD of the Cc:CcO complex. These studies indicate that phosphorylation of these residues plays an important role in regulating mitochondrial electron transport and membrane potential ΔΨ.
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Bakovic, Allison, Nishank Bhalla, Stephanie Kortchak, Chengqun Sun, Weidong Zhou, Aslaa Ahmed, Kenneth Risner, William B. Klimstra, and Aarthi Narayanan. "Venezuelan Equine Encephalitis Virus nsP3 Phosphorylation Can Be Mediated by IKKβ Kinase Activity and Abrogation of Phosphorylation Inhibits Negative-Strand Synthesis." Viruses 12, no. 9 (September 13, 2020): 1021. http://dx.doi.org/10.3390/v12091021.
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Venezuelan equine encephalitis virus (VEEV), a mosquito transmitted alphavirus of the Togaviridae family, can cause a highly inflammatory and encephalitic disease upon infection. Although a category B select agent, no FDA-approved vaccines or therapeutics against VEEV currently exist. We previously demonstrated NF-κB activation and macromolecular reorganization of the IKK complex upon VEEV infection in vitro, with IKKβ inhibition reducing viral replication. Mass spectrometry and confocal microscopy revealed an interaction between IKKβ and VEEV non-structural protein 3 (nsP3). Here, using western blotting, a cell-free kinase activity assay, and mass spectrometry, we demonstrate that IKKβ kinase activity can directly phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5. Alanine substitution mutations at sites 204/5, 142, or 134/5 reduced VEEV replication by >30-100,000-fold corresponding to a severe decrease in negative-strand synthesis. Serial passaging rescued viral replication and negative-strand synthesis, and sequencing of revertant viruses revealed reversion to the wild-type TC-83 phosphorylation capable amino acid sequences at nsP3 sites 204/5, 142, and 135. Generation of phosphomimetic mutants using aspartic acid substitutions at site 204/5 resulted in rescue of both viral replication and negative-strand RNA production, whereas phosphomimetic mutant 134/5 rescued viral replication but failed to restore negative-strand RNA levels, and phosphomimetic mutant 142 did not rescue VEEV replication. Together, these data demonstrate that IKKβ can phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5, and suggest that phosphorylation is essential for negative-strand RNA synthesis at site 204/5, but may be important for infectious particle production at site 134/5.
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Maik-Rachline, Galia, and Rony Seger. "Variable phosphorylation states of pigment-epithelium–derived factor differentially regulate its function." Blood 107, no. 7 (April 1, 2006): 2745–52. http://dx.doi.org/10.1182/blood-2005-06-2547.
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AbstractThe pigment epithelium–derived factor (PEDF) belongs to the family of noninhibitory serpins. Although originally identified in the eye, PEDF is widely expressed in other body regions including the plasma. This factor can act either as a neurotrophic or as an antiangiogenic factor, and we previously showed that the 2 effects of PEDF are regulated through phosphorylation by PKA and CK2. Here, we studied the interplay between the PKA and CK2 phosphorylation of PEDF, and found that a PEDF mutant mimicking the CK2-phosphorylated PEDF cannot be phosphorylated by PKA, while the mutant mimicking the PKA-phosphorylated PEDF is a good CK2 substrate. Using triple mutants that mimic the PKA- and CK2-phosphorylated and nonphosphorylated PEDF, we found that PEDF can induce several distinct cellular activities dependent on its phosphorylation. The mutant mimicking the accumulative PKA plus CK2 phosphorylation exhibited the strongest antiangiogenic and neurotrophic activities, while the mutants mimicking the individual phosphorylation site mutants had either a reduced activity or only one of these activities. Thus, differential phosphorylation induces variable effects of PEDF, and therefore contributes to the complexity of PEDF action. It is likely that the triple phosphomimetic mutant can be used to generate effective antiangiogenic or neurotrophic drugs.
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Deng, Xingming, Fengqin Gao, and W. Stratford May. "Bcl2 retards G1/S cell cycle transition by regulating intracellular ROS." Blood 102, no. 9 (November 1, 2003): 3179–85. http://dx.doi.org/10.1182/blood-2003-04-1027.
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AbstractBcl2's antiapoptotic function is regulated by phosphorylation. Bcl2 also regulates cell cycle progression, but the molecular mechanism is unclear. Bcl2 is functionally expressed in mitochondria where it can act as an antioxidant that may regulate intracellular reactive oxygen species (ROS). Since ROS have been reported to act as second messengers in cell signaling, we tested whether Bcl2 phosphorylation regulates ROS and cell cycle progression. G1 → S transition and ROS levels were measured in cells expressing either the gain of function phosphomimetic Bcl2 mutants S70E and T69E/S70E/S87E (EEE) or the nonphosphorylatable and survival-deficient mutants S70A and T69A/S70A/S87A (AAA). Expression of S70E and EEE but not the A-containing Bcl2 mutants retards G1 → S transition by 35% to 50% and significantly slows cell growth in association with reduced levels of intracellular ROS. In addition to expression of the phosphomimetic Bcl2 mutants, either interleukin-3 withdrawal or treatment of cells with the antioxidant pyrrolidine dithiocarbamate (PDTC) also reduces intracellular ROS levels in association with up-regulation of p27 and accumulation of cells in G0/G1. Retardation of G1 → S transition can be overridden by directly adding H2O2 to the cells in a mechanism that involves down-regulation of p27 and activation of Cdk2. Thus, Bcl2 may regulate G1 → S transition by a novel signaling mechanism that couples regulation of intracellular ROS with p27 and Cdk2. Furthermore, phosphorylation of Bcl2 may functionally link its antiapoptotic, cell cycle retardation, and antioxidant properties.
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Xie, Li, Matthew Kesic, Brenda Yamamoto, Min Li, Ihab Younis, Michael D. Lairmore, and Patrick L. Green. "Human T-Cell Leukemia Virus Type 2 Rex Carboxy Terminus Is an Inhibitory/Stability Domain That Regulates Rex Functional Activity and Viral Replication." Journal of Virology 83, no. 10 (March 11, 2009): 5232–43. http://dx.doi.org/10.1128/jvi.02271-08.
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ABSTRACT Human T-cell leukemia virus (HTLV) regulatory protein, Rex, functions to increase the expression of the viral structural and enzymatic gene products. The phosphorylation of two serine residues (S151 and S153) at the C terminus is important for the function of HTLV-2 Rex (Rex-2). The Rex-2 phosphomimetic double mutant (S151D, S153D) is locked in a functionally active conformation. Since rex and tax genes overlap, Rex S151D and S153D mutants were found to alter the Tax oncoprotein coding sequence and transactivation activities. Therefore, additional Rex-2 mutants including P152D, A157D, S151Term, and S158Term were generated and characterized (“Term” indicates termination codon). All Rex-2 mutants and wild-type (wt) Rex-2 localized predominantly to the nucleus/nucleolus, but in contrast to the detection of phosphorylated and unphosphorylated forms of wt Rex-2 (p26 and p24), mutant proteins were detected as a single phosphoprotein species. We found that Rex P152D, A157D, and S158Term mutants are more functionally active than wt Rex-2 and that the Rex-2 C terminus and its specific phosphorylation state are required for stability and optimal expression. In the context of the provirus, the more active Rex mutants (A157D or S158Term) promoted increased viral protein production, increased viral infectious spread, and enhanced HTLV-2-mediated cellular proliferation. Moreover, these Rex mutant viruses replicated and persisted in inoculated rabbits despite higher antiviral antibody responses. Thus, we identified in Rex-2 a novel C-terminal inhibitory domain that regulates functional activity and is positively regulated through phosphorylation. The ability of this domain to modulate viral replication likely plays a key role in the infectious spread of the virus and in virus-induced cellular proliferation.
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Boeckmann, Lars, Yoshimitsu Takahashi, Wei-Chun Au, Prashant K. Mishra, John S. Choy, Anthony R. Dawson, May Y. Szeto, et al. "Phosphorylation of centromeric histone H3 variant regulates chromosome segregation in Saccharomyces cerevisiae." Molecular Biology of the Cell 24, no. 12 (June 15, 2013): 2034–44. http://dx.doi.org/10.1091/mbc.e12-12-0893.
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The centromeric histone H3 variant (CenH3) is essential for chromosome segregation in eukaryotes. We identify posttranslational modifications of Saccharomyces cerevisiae CenH3, Cse4. Functional characterization of cse4 phosphorylation mutants shows growth and chromosome segregation defects when combined with kinetochore mutants okp1 and ame1. Using a phosphoserine-specific antibody, we show that the association of phosphorylated Cse4 with centromeres increases in response to defective microtubule attachment or reduced cohesion. We determine that evolutionarily conserved Ipl1/Aurora B contributes to phosphorylation of Cse4, as levels of phosphorylated Cse4 are reduced at centromeres in ipl1 strains in vivo, and in vitro assays show phosphorylation of Cse4 by Ipl1. Consistent with these results, we observe that a phosphomimetic cse4-4SD mutant suppresses the temperature-sensitive growth of ipl1-2 and Ipl1 substrate mutants dam1 spc34 and ndc80, which are defective for chromosome biorientation. Furthermore, cell biology approaches using a green fluorescent protein–labeled chromosome show that cse4-4SD suppresses chromosome segregation defects in dam1 spc34 strains. On the basis of these results, we propose that phosphorylation of Cse4 destabilizes defective kinetochores to promote biorientation and ensure faithful chromosome segregation. Taken together, our results provide a detailed analysis, in vivo and in vitro, of Cse4 phosphorylation and its role in promoting faithful chromosome segregation.
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Sun, Kai, Vedrana Montana, Karthikeyani Chellappa, Yann Brelivet, Dino Moras, Yutaka Maeda, Vladimir Parpura, Bryce M. Paschal, and Frances M. Sladek. "Phosphorylation of a Conserved Serine in the Deoxyribonucleic Acid Binding Domain of Nuclear Receptors Alters Intracellular Localization." Molecular Endocrinology 21, no. 6 (June 1, 2007): 1297–311. http://dx.doi.org/10.1210/me.2006-0300.
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Abstract Nuclear receptors (NRs) are a superfamily of transcription factors whose genomic functions are known to be activated by lipophilic ligands, but little is known about how to deactivate them or how to turn on their nongenomic functions. One obvious mechanism is to alter the nuclear localization of the receptors. Here, we show that protein kinase C (PKC) phosphorylates a highly conserved serine (Ser) between the two zinc fingers of the DNA binding domain of orphan receptor hepatocyte nuclear factor 4α (HNF4α). This Ser (S78) is adjacent to several positively charged residues (Arg or Lys), which we show here are involved in nuclear localization of HNF4α and are conserved in nearly all other NRs, along with the Ser/threonine (Thr). A phosphomimetic mutant of HNF4α (S78D) reduced DNA binding, transactivation ability, and protein stability. It also impaired nuclear localization, an effect that was greatly enhanced in the MODY1 mutant Q268X. Treatment of the hepatocellular carcinoma cell line HepG2 with PKC activator phorbol 12-myristate 13-acetate also resulted in increased cytoplasmic localization of HNF4α as well as decreased endogenous HNF4α protein levels in a proteasome-dependent fashion. We also show that PKC phosphorylates the DNA binding domain of other NRs (retinoic acid receptor α, retinoid X receptor α, and thyroid hormone receptor β) and that phosphomimetic mutants of the same Ser/Thr result in cytoplasmic localization of retinoid X receptor α and peroxisome proliferator-activated receptor α. Thus, phosphorylation of this conserved Ser between the two zinc fingers may be a common mechanism for regulating the function of NRs.
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Matsumoto, Tadahiko, Kotaro Shirakawa, Hiroyuki Matsui, Hiroyuki Yamazaki, Yasuhiro Kazuma, Anamaria Daniela Sarca, Hirofumi Fukuda, Wataru Maruyama, and Akifumi Takaori-Kondo. "PKA-Mediated Phosphorylation of APOBEC3B Suppresses Its DNA Mutagenic Potential in Myeloma Cells." Blood 128, no. 22 (December 2, 2016): 4427. http://dx.doi.org/10.1182/blood.v128.22.4427.4427.
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Abstract Multiple myeloma (MM) is an incurable plasma cell malignancy and the reason for this is not yet completely understood. It might be partially attributable to heterogeneity and clonal evolution through DNA mutation accumulation. Recent next generation sequencing (NGS) studies have revealed that apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) 3B (A3B) is one of the cause of clonal evolution in MM. APOBEC protein family consists of 11 proteins and most of them, including A3B, have cytidine deaminase (CDA) activity on single-strand DNA, which catalyzes deamination of cytosine to uridine, resulting in C-to-T or C-to-G mutations. We have previously shown in human cell line models that A3B induces genomic mutations (Shinohara et al., Sci Rep, 2012). Therefore, controlling A3B CDA activity is an attractive therapeutic strategy to overcome the unfavorable prognosis of patients with these malignancies. Enzymatic activity of other APBEC family proteins, activation-induced cytidine deaminase (AID) and APOBEC3G (A3G) is regulated by protein kinase A (PKA)-mediated phosphorylation. A3B also has two consensus PKA phosphorylation motifs conserved upstream of each CDA domain. Thus, we hypothesized that PKA phosphorylates A3B which may alter A3B CDA activity. Using NetPhosK1.0 and ScanSite programs, two A3B residues (serine at the 46th and threonine at the 214th position) are predicted to be PKA phosphorylation sites. Firstly, we examined the binding between A3B and PKA. We transfected HEK293T cells with expression vectors for C-terminal HA-tagged A3B and N-terminal FLAG-tagged PKA catalytic subunit a (PKACA) and used CoIP assays with anti-HA or anti-FLAG antibodies to demonstrate physical binding between A3B and PKACA. Secondly, to determine whether the putative residues are phosphorylated, we overexpressed PKACA and A3B alanine mutants in HEK293T cells and performed immunoblotting analysis with anti-phospho PKA motif antibodies and found that threonine-214 is phosphorylated by PKA. To confirm that PKA phosphorylates A3B directly, we employed in vitro phosphorylation assays using purified C-terminal A3B and PKACA and found that PKACA phosphorylated A3B, but not the A3B T214A mutant, thus we concluded that PKACA directly phosphorylates threonine-214 in A3B. Next, to investigate the effect of this phosphorylation on the CDA activity of A3B, we constructed phosphomimetic mutants of these residues, T214D and T214E. We carried out in vitro CDA assays using cell lysates from HEK293T cells overexpressing A3B or mutants (T214A, T214D, T214E), and found that T214D and T214E almost completely lost their CDA activity. We also performed in vitro CDA assays using purified C-terminal A3B and mutant proteins. The purified A3B T214D mutant also lost CDA activity, suggesting that PKA-mediated phosphorylation of A3B might also lead to loss of its CDA activity. To confirm this result in cell lines, we used differential DNA denaturation PCR (3D-PCR) of DNA from HEK293T cells transfected with vectors for uracil DNA glycosylase inhibitor, EGFP and A3B wild type or mutants. 3D-PCR products of the EGFP sequence were amplified at a lower denaturation temperature in cells expressing the wild type A3B compared to those in cells expressing phosphomimetic A3B or catalytically inactivated A3B. These results indicate that phosphomimetic A3B no longer has exogenous DNA editing activity. We further plan to evaluate the PKA phosphorylation of A3B and the consequences on its mutagenic potential in myeloma cell lines. In conclusion, PKA induces phosphorylation at threonine-214 in A3B, and this phosphorylation suppresses its CDA activity. PKA-mediated phosphorylation of A3B may be an attractive therapeutic target for inhibiting A3B-induced clonal evolution in lymphoid malignancies, especially in MM. Disclosures Takaori-Kondo: Kyowa Kirin: Research Funding; Alexion Pharmaceuticals: Research Funding; Eisai: Research Funding; Shionogi: Research Funding; Chugai Pharmaceutical: Research Funding; Mochida Pharmaceutical: Research Funding; Takeda Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Pfizer: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Merck Sharp and Dohme: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Toyama Chemical: Research Funding; Cognano: Research Funding.
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Hu, Yifan, Kavita Iyer, Ashok R. Nayak, Thomas Klose, Jose M. Eltit, Nagomi Kurebayashi, Takashi Murayama, and Montserrat Samso. "Cryo-Em Studies of Phosphomimetic and Phospho-Null Triple Mutants of Cardiac Ryanodine Receptor (RyR2)." Biophysical Journal 120, no. 3 (February 2021): 239a. http://dx.doi.org/10.1016/j.bpj.2020.11.1574.
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Konson, Alexander, Sunila Pradeep, and Rony Seger. "Phosphomimetic Mutants of Pigment Epithelium-Derived Factor with Enhanced Antiangiogenic Activity as Potent Anticancer Agents." Cancer Research 70, no. 15 (July 7, 2010): 6247–57. http://dx.doi.org/10.1158/0008-5472.can-10-0434.
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Effenberger, Madlen, Valentin Bruttel, Ralf C. Bargou, and Kurt Bommert. "The Relevance of Phosphorylated YB-1 for Multiple Myeloma Cell Survival and Drug Resistance." Blood 112, no. 11 (November 16, 2008): 734. http://dx.doi.org/10.1182/blood.v112.11.734.734.
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Abstract Y-Box protein 1 (YB-1) belongs to the evolutionarily highly conserved family of cold-shock domain proteins. YB-1 binds to DNA as well as RNA and fulfils pleiotropic cellular functions, including transcriptional and translational regulation of proteins involved in cellular growth, survival and stress response. YB-1 over-expression can be detected in a variety of human cancers and, if located in the nucleus, YB-1 over-expression correlates with drug-resistance and an unfavorable clinical outcome. In transgenic mice, mammary gland specific over expression of human YB 1 provokes breast cancer with a 100 % penetrance. YB-1 knockout experiments in mice showed that a homozygous deletion is lethal and a heterozygous YB 1 deletion is accompanied with an increased sensitivity to cisplatin and mitomycin C. Immunohistochemical analysis of 41 human multiple myeloma (MM) samples showed that YB-1 is strongly expressed in a highly proliferative subset of primary MM cells which are characterized by Ki67 staining and an immature morphology. The cellular localization of YB-1 may determine if YB-1 has an oncogenic or anti-oncogenic function. Nuclear YB-1 regulates transcription of genes involved in cell proliferation and differentiation. Cytoplasmic YB-1 is involved in packaging mRNAs into translational inactive messenger ribonucleoprotein particles (mRNPs) and attaching them to microtubules. Akt mediated phosphorylation of YB-1 at S102 disables the binding of YB-1 to mRNAs and leads to the translocation of YB-1 into the nucleus. The nuclear localization is accompanied with an aggressive phenotype and increased apoptosis resistance, representing the oncogenic function of YB-1. In contrast the non-phosphorylatable YB-1 mutant (S102A) remains in the cytoplasm bound to the cap structure of mRNAs and mediates the anti-oncogenic function. To characterize the significance of the strong YB-1 expression in MM we generated YB-1 mutants with an additional C-terminal HaloTag®. We labeled the tag with a fluorescent ligand to monitor the subcellular localization of the YB-1 mutants in living MM cell lines using confocal microscopy. Here we show that the phosphomimetic (S102E) YB-1 mutant is located preferentially in the nucleus, whereas the non-phosphorylatable (S102A) mutant is cytoplasmic in MM cell lines. These mutants were analyzed for the functional significance of the phosphorylation on S102 in MM cell lines. Cells over-expressing the phosphomimetic YB-1 (S102E) are more viable and resistant to doxorubicin treatment compared to YB-1 wild-type and S102A transfected cells. These results suggest that phosphorylation and nuclear localization of YB-1 are essential for tumor cell survival and drug resistance in MM. The phenotypic change seen after translocation of YB-1 to the nucleus can be mediated either by the translation of previously inhibited mRNAs, by nuclear YB-1 initiated gene transcription, or by a mixture of both. Here we present the data comparing mRNAs bound to the different YB-1 mutants and the corresponding gene expression profiles in different MM cell lines.
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Yang, Yan, Tim J. Craig, Xiaohui Chen, Leonora F. Ciufo, Masami Takahashi, Alan Morgan, and Kevin D. Gillis. "Phosphomimetic Mutation of Ser-187 of SNAP-25 Increases both Syntaxin Binding and Highly Ca2+-sensitive Exocytosis." Journal of General Physiology 129, no. 3 (February 26, 2007): 233–44. http://dx.doi.org/10.1085/jgp.200609685.
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The phosphorylation targets that mediate the enhancement of exocytosis by PKC are unknown. PKC phosporylates the SNARE protein SNAP-25 at Ser-187. We expressed mutants of SNAP-25 using the Semliki Forest Virus system in bovine adrenal chromaffin cells and then directly measured the Ca2+ dependence of exocytosis using photorelease of caged Ca2+ together with patch-clamp capacitance measurements. A flash of UV light used to elevate [Ca2+]i to several μM and release the highly Ca2+-sensitive pool (HCSP) of vesicles was followed by a train of depolarizing pulses to elicit exocytosis from the less Ca2+-sensitive readily releasable pool (RRP) of vesicles. Carbon fiber amperometry confirmed that the amount and kinetics of catecholamine release from individual granules were similar for the two phases of exocytosis. Mimicking PKC phosphorylation with expression of the S187E SNAP-25 mutant resulted in an approximately threefold increase in the HCSP, whereas the response to depolarization increased only 1.5-fold. The phosphomimetic S187D mutation resulted in an ∼1.5-fold increase in the HCSP but a 30% smaller response to depolarization. In vitro binding assays with recombinant SNARE proteins were performed to examine shifts in protein–protein binding that may promote the highly Ca2+-sensitive state. The S187E mutant exhibited increased binding to syntaxin but decreased Ca2+-independent binding to synaptotagmin I. Mimicking phosphorylation of the putative PKA phosphorylation site of SNAP-25 with the T138E mutation decreased binding to both syntaxin and synaptotagmin I in vitro. Expressing the T138E/ S187E double mutant in chromaffin cells demonstrated that enhancing the size of the HCSP correlates with an increase in SNAP-25 binding to syntaxin in vitro, but not with Ca2+-independent binding of SNAP-25 to synaptotagmin I. Our results support the hypothesis that exocytosis triggered by lower Ca2+ concentrations (from the HCSP) occurs by different molecular mechanisms than exocytosis triggered by higher Ca2+ levels.
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Thévenin, Anastasia F., Rachel A. Margraf, Charles G. Fisher, Rachael M. Kells-Andrews, and Matthias M. Falk. "Phosphorylation regulates connexin43/ZO-1 binding and release, an important step in gap junction turnover." Molecular Biology of the Cell 28, no. 25 (December 2017): 3595–608. http://dx.doi.org/10.1091/mbc.e16-07-0496.
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To investigate whether connexin phosphorylation regulates the known role of zonula occludens-1 protein (ZO-1) in gap junction (GJ) function, we generated and analyzed a series of phosphomimetic and phosphorylation-dead mutants by mutating known conserved regulatory serine (S) residues 255, 279/282, 365, 368, and 373 located in the C-terminal domain of connexin43 (Cx43) into glutamic acid (E) or alanine (A) residues. All connexin mutants were translated into stable, full-length proteins and assembled into GJs when expressed in HeLa or Madin–Darby canine kidney epithelial cells. However, mutants with S residues exchanged at positions 365, 368, and 373 exhibited a significantly altered ZO-1 interaction profile, while mutants with S residues exchanged at 255 and 279/282 did not. Unlike wild-type Cx43, in which ZO-1 binding is restricted to the periphery of GJ plaques, S365A, S365E, S368A, S368E, and S373A mutants bound ZO-1 throughout the GJ plaques, while the S373E mutant did not bind ZO-1 at all. Inability to disengage from ZO-1 correlated with increased GJ plaque size and increased connexin protein half-life, while maintaining GJ channels in an open, functional state. Quantitative clathrin-binding analyses revealed no significant alterations in clathrin-binding efficiency, suggesting that the inability to disengage from ZO-1 prevented maturation of functional into nonfunctional/endocytic channels, rather than ZO-1 interfering with GJ endocytosis directly. Collectively, our results indicate that ZO-1 binding regulates channel accrual, while disengagement from ZO-1 is critical for GJ channel closure and transitioning GJ channels for endocytosis. Intriguingly, these transitional ZO-1 binding/release and channel-aging steps are mediated by a series of hierarchical phosphorylation/dephosphorylation events at S373, S365, and S368, well-known Cx43 Akt, protein kinase A, and protein kinase C phosphorylation sites located in the vicinity of the ZO-1 binding site.
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Mishra, Neeraj Kumar, Michael Habeck, Corinna Kirchner, Haim Haviv, Yoav Peleg, Miriam Eisenstein, Hans Juergen Apell, and Steven J. D. Karlish. "Molecular Mechanisms and Kinetic Effects of FXYD1 and Phosphomimetic Mutants on Purified Human Na,K-ATPase." Journal of Biological Chemistry 290, no. 48 (October 1, 2015): 28746–59. http://dx.doi.org/10.1074/jbc.m115.687913.
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Guerra-Castellano, Alejandra, Irene Díaz-Moreno, Adrián Velázquez-Campoy, Miguel A. De la Rosa, and Antonio Díaz-Quintana. "Structural and functional characterization of phosphomimetic mutants of cytochrome c at threonine 28 and serine 47." Biochimica et Biophysica Acta (BBA) - Bioenergetics 1857, no. 4 (April 2016): 387–95. http://dx.doi.org/10.1016/j.bbabio.2016.01.011.
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Katoshevski, Tomer, Lior Bar, Eliav Tikochinsky, Shimon Harel, Tsipi Ben-Kasus Nissim, Ivan Bogeski, Michal Hershfinkel, Bernard Attali, and Israel Sekler. "CKII Control of Axonal Plasticity Is Mediated by Mitochondrial Ca2+ via Mitochondrial NCLX." Cells 11, no. 24 (December 9, 2022): 3990. http://dx.doi.org/10.3390/cells11243990.
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Mitochondrial Ca2+ efflux by NCLX is a critical rate-limiting step in mitochondria signaling. We previously showed that NCLX is phosphorylated at a putative Casein Kinase 2 (CKII) site, the serine 271 (S271). Here, we asked if NCLX is regulated by CKII and interrogated the physiological implications of this control. We found that CKII inhibitors down-regulated NCLX-dependent Ca2+ transport activity in SH-SY5Y neuronal cells and primary hippocampal neurons. Furthermore, we show that the CKII phosphomimetic mutants on NCLX inhibited (S271A) and constitutively activated (S271D) NCLX transport, respectively, rendering it insensitive to CKII inhibition. These phosphomimetic NCLX mutations also control the allosteric regulation of NCLX by mitochondrial membrane potential (ΔΨm). Since the omnipresent CKII is necessary for modulating the plasticity of the axon initial segment (AIS), we interrogated, in hippocampal neurons, if NCLX is required for this process. Similarly to WT neurons, NCLX-KO neurons can exhibit homeostatic plasticity following M-channel block. However, while WT neurons utilize a CKII-sensitive distal relocation of AIS Na+ and Kv7 channels to decrease their intrinsic excitability, we did not observe such translocation in NCLX-KO neurons. Thus, our results indicate that NCLX is regulated by CKII and is a crucial link between CKII signaling and fast neuronal plasticity.
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Hao, Jian-Jiang, Yin Liu, Michael Kruhlak, Karen E. Debell, Barbara L. Rellahan, and Stephen Shaw. "Phospholipase C–mediated hydrolysis of PIP2 releases ERM proteins from lymphocyte membrane." Journal of Cell Biology 184, no. 3 (February 9, 2009): 451–62. http://dx.doi.org/10.1083/jcb.200807047.
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Mechanisms controlling the disassembly of ezrin/radixin/moesin (ERM) proteins, which link the cytoskeleton to the plasma membrane, are incompletely understood. In lymphocytes, chemokine (e.g., SDF-1) stimulation inactivates ERM proteins, causing their release from the plasma membrane and dephosphorylation. SDF-1–mediated inactivation of ERM proteins is blocked by phospholipase C (PLC) inhibitors. Conversely, reduction of phosphatidylinositol 4,5-bisphosphate (PIP2) levels by activation of PLC, expression of active PLC mutants, or acute targeting of phosphoinositide 5-phosphatase to the plasma membrane promotes release and dephosphorylation of moesin and ezrin. Although expression of phosphomimetic moesin (T558D) or ezrin (T567D) mutants enhances membrane association, activation of PLC still relocalizes them to the cytosol. Similarly, in vitro binding of ERM proteins to the cytoplasmic tail of CD44 is also dependent on PIP2. These results demonstrate a new role of PLCs in rapid cytoskeletal remodeling and an additional key role of PIP2 in ERM protein biology, namely hydrolysis-mediated ERM inactivation.
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Jewell, Jenna L., Eunjin Oh, Latha Ramalingam, Michael A. Kalwat, Vincent S. Tagliabracci, Lixuan Tackett, Jeffrey S. Elmendorf, and Debbie C. Thurmond. "Munc18c phosphorylation by the insulin receptor links cell signaling directly to SNARE exocytosis." Journal of Cell Biology 193, no. 1 (March 28, 2011): 185–99. http://dx.doi.org/10.1083/jcb.201007176.
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How the Sec1/Munc18–syntaxin complex might transition to form the SNARE core complex remains unclear. Toward this, Munc18c tyrosine phosphorylation has been correlated with its dissociation from syntaxin 4. Using 3T3-L1 adipocytes subjected to small interfering ribonucleic acid reduction of Munc18c as a model of impaired insulin-stimulated GLUT4 vesicle exocytosis, we found that coordinate expression of Munc18c–wild type or select phosphomimetic Munc18c mutants, but not phosphodefective mutants, restored GLUT4 vesicle exocytosis, suggesting a requirement for Munc18c tyrosine phosphorylation at Tyr219 and Tyr521. Surprisingly, the insulin receptor (IR) tyrosine kinase was found to target Munc18c at Tyr521 in vitro, rapidly binding and phosphorylating endogenous Munc18c within adipocytes and skeletal muscle. IR, but not phosphatidylinositol 3-kinase, activation was required. Altogether, we identify IR as the first known tyrosine kinase for Munc18c as part of a new insulin-signaling step in GLUT4 vesicle exocytosis, exemplifying a new model for the coordination of SNARE assembly and vesicle mobilization events in response to a single extracellular stimulus.
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Sevcovicova, Andrea, Jana Plava, Matej Gazdarica, Eva Szabova, Barbora Huraiova, Katarina Gaplovska-Kysela, Ingrid Cipakova, Lubos Cipak, and Juraj Gregan. "Mapping and Analysis of Swi5 and Sfr1 Phosphorylation Sites." Genes 12, no. 7 (June 30, 2021): 1014. http://dx.doi.org/10.3390/genes12071014.
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The evolutionarily conserved Swi5-Sfr1 complex plays an important role in homologous recombination, a process crucial for the maintenance of genomic integrity. Here, we purified Schizosaccharomyces pombe Swi5-Sfr1 complex from meiotic cells and analyzed it by mass spectrometry. Our analysis revealed new phosphorylation sites on Swi5 and Sfr1. We found that mutations that prevent phosphorylation of Swi5 and Sfr1 do not impair their function but swi5 and sfr1 mutants encoding phosphomimetic aspartate at the identified phosphorylation sites are only partially functional. We conclude that during meiosis, Swi5 associates with Sfr1 and both Swi5 and Sfr1 proteins are phosphorylated. However, the functional relevance of Swi5 and Sfr1 phosphorylation remains to be determined.
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Hu, Yifan, Kavita A. Iyer, Ashok R. Nayak, Jose M. Eltit, Nagomi Kurebayashi, Takashi Murayama, and Montserrat Samso. "Phosphorylation of cardiac ryanodine receptor (RyR2): a structure-function study of phosphomimetic and phospho-null triple mutants." Biophysical Journal 121, no. 3 (February 2022): 379a. http://dx.doi.org/10.1016/j.bpj.2021.11.867.
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Baliova, Martina, and Frantisek Jursky. "Correlation of calpain sensitivity, Bradford assay instability, and electrophoretic mobility in phosphomimetic mutants of GlyT2 N-terminus." Biochemistry and Biophysics Reports 38 (July 2024): 101734. http://dx.doi.org/10.1016/j.bbrep.2024.101734.
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Narayan, Murli, Ihab Younis, Donna M. D'Agostino, and Patrick L. Green. "Functional Domain Structure of Human T-Cell Leukemia Virus Type 2 Rex." Journal of Virology 77, no. 23 (December 1, 2003): 12829–40. http://dx.doi.org/10.1128/jvi.77.23.12829-12840.2003.
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ABSTRACT The Rex protein of human T-cell leukemia virus (HTLV) acts posttranscriptionally to induce the cytoplasmic expression of the unspliced and incompletely spliced viral RNAs encoding the viral structural and enzymatic proteins and is therefore essential for efficient viral replication. Rex function requires nuclear import, RNA binding, multimerization, and nuclear export. In addition, it has been demonstrated that the phosphorylation status of HTLV-2 Rex (Rex-2) correlates with RNA binding and inhibition of splicing in vitro. Recent mutational analyses of Rex-2 revealed that the phosphorylation of serine residues 151 and 153 within a novel carboxy-terminal domain is critical for function in vivo. To further define the functional domain structure of Rex-2, we evaluated a panel of Rex-2 mutants for subcellular localization, RNA binding capacity, multimerization and trans-dominant properties, and the ability to shuttle between the nucleus and the cytoplasm. Rex-2 mutant S151A,S153A, which is defective in phosphorylation and function, showed diffuse cytoplasmic staining, whereas mutant S151D,S153D, previously shown to be functional and in a conformation corresponding to constitutive phosphorylation, displayed increased intense speckled staining in the nucleoli. In vivo RNA binding analyses indicated that mutant S151A,S153A failed to efficiently bind target RNA, while its phosphomimetic counterpart, S151D,S153D, bound twofold more RNA than wild-type Rex-2. Taken together, these findings provide direct evidence that the phosphorylation status of Rex-2 is linked to cellular trafficking and RNA binding capacity. Mutants with substitutions in either of the two putative multimerization domains or in the putative activation domain-nuclear export signal displayed a dominant negative phenotype as well as defects in multimerization and nucleocytoplasmic shuttling. Several carboxy-terminal mutants that displayed wild-type levels of phosphorylation and localized to the nucleolus were also partially impaired in shuttling. This is consistent with the hypothesis that the carboxy terminus of Rex-2 contains a novel domain that is required for efficient shuttling. This work thus provides a more detailed functional domain map of Rex-2 and further insight into its regulation of HTLV replication.
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Andreeff, Michael, Rooha Contractor, Peter P. Ruvolo, Xingming Deng, Ismael Samudio, Yue-Xi Shi, Teresa McQueen, et al. "Mechanisms of Apoptosis Induction by BH3 Inhibitor ABT-737 in AML." Blood 106, no. 11 (November 16, 2005): 244. http://dx.doi.org/10.1182/blood.v106.11.244.244.
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Abstract Bcl2 family proteins are key regulators of apoptosis. Aberrations in Bcl2 levels are known to promote tumorigenesis and chemoresistance. Thus, strategies to target Bcl2 will likely provide effective therapies for malignancies such as acute myeloid leukemia (AML). In this report, we investigate mechanisms of action of the novel small molecule Bcl2 inhibitor ABT-737 in AML. ABT-737 effectively killed AML patient blast cells and colony-forming cell lines at nanomolar concentrations with no effect on normal hematopoietic cells. Notably, CD34+38−123+ AML stem cells are highly sensitive to the compound. ABT-737-induced apoptosis is initiated by disruption of Bcl2:Bax dimers and activation of the intrinsic apoptotic pathway. ABT-737 works synergistically with chemotherapeutic agents such as ara-C and doxorubicin. To investigate the role of Bcl-2 phosphorylation in the sensitivity to BH3 inhibitor, we used IL-3 dependent NSF.N1/H7 mouse myeloid cells modified by site-directed mutagenesis to produce various Bcl-2 phospho-mutants. NSF.N1/H7 cells stably transfected with phosphomimetic T69E/S70E/S87E (EEE) Bcl-2 mutants were resistant to ABT-737 (IC50>500 nM) as compared to cells expressing wt-Bcl-2 or the nonphosphorylatable T69A/S70A/S87A (AAA) Bcl2 mutants (IC50s of 50 and 25 nM). Consistent with a mechanism whereby increased Bcl2 phosphorylation impedes ABT-737 suppression of Bcl2 dimerization with Bax, ABT-737 potently blocked Bcl2:Bax association in cells expressing exogenous WT Bcl2 and AAA mutant Bcl2 but not in cells expressing exogenous phosphomimetic EEE mutant Bcl2. Since the S70E phosphorylation site of Bcl-2 is a known ERK substrate, we examined combined effects of ABT-737 and MEK inhibitor PD98059 in OCI-AML3 cells resistant to ABT-737 alone. The combined activity of PD98059 and ABT-737, evaluated by isobologram analysis, revealed a striking synergistic interaction between the MEK and BH3 inhibitors, with combination indices (CI) of 0.08±0.003. OCI-AML3 cells exhibit the highest expression of Mcl-1 among the acute leukemia cell lines tested. We propose that loss of Mcl-1 expression as a result of suppression of ERK may also be involved in the ability of PD98059 to enhance ABT-737-induced apoptosis. siRNA to Mcl-1 strikingly sensitized OCI-AML3 cells to ABT-induced apoptosis (14% apoptosis in parental cells at 2.5μM ABT-737, 64% apoptosis in siRNA-transfected cells at 10-fold lower concentration of 0.25μM). We have further demonstrated that ABT-737 reduced leukemia burden and significantly (p=0.0018) prolonged survival of mice in an in vivo mouse model. These findings suggest that: 1) ABT-737 reduces apoptosis through disruption of Bcl2:Bax heterodimers; 2) its activity is limited by Bcl2 phosphorylation and Mcl-1 overexpression; 3) combination with MEK inhibition results in inhibition of Bcl2 phosphorylation, downregulation of Mcl-1 and dramatic enhancement of ABT-737-induced apoptosis in AML.
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Sidorenko, Viktoriya S., Arthur P. Grollman, Pawel Jaruga, Miral Dizdaroglu, and Dmitry O. Zharkov. "Substrate specificity and excision kinetics of natural polymorphic variants and phosphomimetic mutants of human 8-oxoguanine-DNA glycosylase." FEBS Journal 276, no. 18 (August 7, 2009): 5149–62. http://dx.doi.org/10.1111/j.1742-4658.2009.07212.x.
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Liu, Yuan, Yao Wei, Chaoyun Pan, Dihan Zhu, Lei Shi, Zhen Bian, and Ke Zen. "Pyruvate kinase type M2 promotes tumor cell exocytosis via phosphorylating synaptosome-associated protein-23." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 72.14. http://dx.doi.org/10.4049/jimmunol.196.supp.72.14.
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Abstract Tumor cells secret large amount of microvesicles (MVs) which are involved in remodeling tumor-stromal interactions to promote malignancy. The regulatory mechanisms governing the active exocytosis in tumor cells, however, remain incompletely understood. We show here that tumor cell exocytosis is tightly controlled by aerobic glycolysis (Warburg effect) and pyruvate kinase type M2 (PKM2), which is commonly upregulated and phosphorylated in tumor cells, plays critical roles in promoting exocytosis. Upon stimulation by EGF, more phosphorylated PKM2 switches to dimer form and is recruited to exosomes where it associates with synaptosome-associated protein (SNAP)-23 (SNAP-23). The association of PKM2 and SNAP-23 in secreted exosomes is confirmed by cross immunoprecipitation and Western blot analysis. Dimerized PKM2 enhances the exosome release via phosphorylating SNAP-23. Direct phosphorylation assay and mass spectrometry identifies SNAP-23 phosphorylation at Ser95by PKM2. Ectopic expression of non-phosphorylated SNAP-23 mutant (Ser95→Ala95) significantly reduces PKM2-mediated exocytosis whereas phosphomimetic SNAP-23 mutants (Ser95→Ele95) partially rescue the impaired exocytosis in tumor cells with PKM2 knockdown. Our findings reveal a previously unrecognized non-metabolic function of PKM2 in promoting tumor cell exocytosis.
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Jacobsen, Nicole L., Tasha K. Pontifex, Paul R. Langlais, and Janis M. Burt. "Phosphorylation-Dependent Intra-Domain Interaction of the Cx37 Carboxyl-Terminus Controls Cell Survival." Cancers 11, no. 2 (February 6, 2019): 188. http://dx.doi.org/10.3390/cancers11020188.
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Differential phosphorylation of the carboxyl-terminus of connexin 37 (Cx37-CT) regulates phenotypic switching between cell growth phenotypes (cell death, cell cycle arrest, proliferation). The specific phosphorylation events in the Cx37-CT that are necessary for these growth regulatory effects are currently unknown. Through the combined use of deletion and site specific (de)phospho-mimetic Cx37-CT mutants, our data suggest a phosphorylation-dependent interaction between the mid-tail (aa 273–317) and end-tail (aa 318–333) portions of the Cx37-CT that regulates cell survival. As detected by mass spectrometry, Cx37 was phosphorylated at serines 275, 321, and 328; phosphomimetic mutations of these sites resulted in cell death when expressed in rat insulinoma cells. Alanine substitution at S328, but not at S275 or S321, also triggered cell death. Cx37-S275D uniquely induced the death of only low density, non-contact forming cells, but neither hemichannel open probability nor channel conductance distinguished death-inducing mutants. As channel function is necessary for cell death, together the data suggest that the phosphorylation state of the Cx37-CT controls an intra-domain interaction within the CT that modifies channel function and induces cell death.
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Wagner, Larry E., Wen-Hong Li, Suresh K. Joseph, and David I. Yule. "Functional Consequences of Phosphomimetic Mutations at Key cAMP-dependent Protein Kinase Phosphorylation Sites in the Type 1 Inositol 1,4,5-Trisphosphate Receptor." Journal of Biological Chemistry 279, no. 44 (August 11, 2004): 46242–52. http://dx.doi.org/10.1074/jbc.m405849200.
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Regulation of Ca2+release through inositol 1,4,5-trisphosphate receptors (InsP3R) has important consequences for defining the particular spatio-temporal properties of intracellular Ca2+signals. In this study, regulation of Ca2+release by phosphorylation of type 1 InsP3R (InsP3R-1) was investigated by constructing “phosphomimetic” charge mutations in the functionally important phosphorylation sites of both the S2+ and S2- InsP3R-1 splice variants. Ca2+release was investigated following expression in Dt-40 3ko cells devoid of endogenous InsP3R. In cells expressing either the S1755E S2+ or S1589E/S1755E S2- InsP3R-1, InsP3-induced Ca2+release was markedly enhanced compared with nonphosphorylatable S2+ S1755A and S2- S1589A/S1755A mutants. Ca2+release through the S2- S1589E/S1755E InsP3R-1 was enhanced ∼8-fold over wild type and ∼50-fold when compared with the nonphosphorylatable S2- S1589A/S1755A mutant. In cells expressing S2- InsP3R-1 with single mutations in either S1589E or S1755E, the sensitivity of Ca2+release was enhanced ∼3-fold; sensitivity was midway between the wild type and the double glutamate mutation. Paradoxically, forskolin treatment of cells expressing either single Ser/Glu mutation failed to further enhance Ca2+release. The sensitivity of Ca2+release in cells expressing S2+ S1755E InsP3R-1 was comparable with the sensitivity of S2- S1589E/S1755E InsP3R-1. In contrast, mutation of S2+ S1589E InsP3R-1 resulted in a receptor with comparable sensitivity to wild type cells. Expression of S2- S1589E/S1755E InsP3R-1 resulted in robust Ca2+oscillations when cells were stimulated with concentrations of α-IgM antibody that were threshold for stimulation in S2- wild type InsP3R-1-expressing cells. However, at higher concentrations of α-IgM antibody, Ca2+oscillations of a similar period and magnitude were initiated in cells expressing either wild type or S2- phosphomimetic mutations. Thus, regulation by phosphorylation of the functional sensitivity of InsP3R-1 appears to define the threshold at which oscillations are initiated but not the frequency or amplitude of the signal when established.
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Macari, Elizabeth R., Alison Taylor, David Raiser, Kavitha Siva, Katherine McGrath, Jessica M. Humphries, Johan Flygare, Benjamin L. Ebert, and Leonard I. Zon. "Calmodulin Inhibition Rescues the Effects of Ribosomal Protein Deficiency in in Vitro and In Vivo Diamond Blackfan Anemia Models." Blood 126, no. 23 (December 3, 2015): 672. http://dx.doi.org/10.1182/blood.v126.23.672.672.
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Abstract Ribosomal protein (RP) mutations are found in many diseases, including Diamond Blackfan anemia (DBA), where defective erythropoiesis, craniofacial abnormalities and increased cancer risk are major complications. RP mutations cause p53 activation through accumulation of free RPs that bind and sequester MDM2, the negative regulator of p53. We previously characterized a zebrafish mutant in rps29, a gene found mutated in DBA patients. Rps29-/- embryos have hematopoietic and endothelial defects, including decreased cmyb and flk1 expression and defects in hemoglobinization. Consistent with other animal models of RP dysfunction, p53 knockdown in rps29-/- embryos rescued these defects. To uncover novel compounds that correct the phenotypes of DBA, we performed a chemical screen in rps29-/- embryos. Several structurally distinct calmodulin (CaM) inhibitors successfully rescued hemoglobin (Hb) levels in the mutant embryo. To confirm that CaM inhibitors could rescue mammalian models of DBA, we applied them to human and murine models. Treating cord blood-derived CD34+ cells deficient in RPS19 with the CaM inhibitor, trifluoperazine (TFP), relieved the erythroid differentiation block. Injection of TFP in a DBA murine model significantly increased red blood cell number and Hb levels. Mechanistic studies in A549 cells infected with lentivirus expressing RPS19 shRNA demonstrated that TFP blocks p53 nuclear accumulation and induction of multiple p53 transcriptional target genes (p<0.05). Through p53 genetic manipulation, we determined that TFP inhibits p53 transcriptional activity through its c-terminal domain (CTD). Since this region has many residues that can be phosphorylated by CaM-dependent kinases, we hypothesized that TFP blocked phosphorylation of residues in the CTD. To test this hypothesis, phosphomimetic mutants were transfected into Saos2 cells and p53 transcriptional activity in response to TFP was evaluated using p21mRNA levels. TFP treatment of cells containing WT p53 or a transactivation domain mutant, S15D, resulted in a 4-fold reduction in p21 mRNA levels, while all four phosphomimetic mutants in the CTD had attenuated responses to TFP (<2-fold). The common CaM-dependent kinases that phosphorylate these CTD residues are Chk1 and Chk2. Investigation of the role of Chk1 and Chk2 found that a chk2 morpholino and multiple inhibitors of Chk2, but not Chk1, rescued Hb levels in the rps29-/- embryo (p<0.05). Chk2 inhibitors also mimic CaM inhibition in our in vitro assays. In conclusion, we have shown a novel mechanism by which CaM inhibitors mediate p53 activity through the CTD and can rescue the phenotypes of multiple in vitro and in vivo models of DBA. Our data strongly suggests that CaM or Chk2 inhibitors may be effective therapies for DBA patients, and a clinical trial is being planned with TFP. Disclosures Ebert: Genoptix: Consultancy, Patents & Royalties; H3 Biomedicine: Consultancy; Celgene: Consultancy. Zon:FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.
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Morimoto, Hiroko, Kunio Kondoh, Satoko Nishimoto, Kazuya Terasawa, and Eisuke Nishida. "Activation of a C-terminal Transcriptional Activation Domain of ERK5 by Autophosphorylation." Journal of Biological Chemistry 282, no. 49 (October 10, 2007): 35449–56. http://dx.doi.org/10.1074/jbc.m704079200.
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ERK5 plays a crucial role in many biological processes by regulating transcription. ERK5 has a large C-terminal-half that contains a transcriptional activation domain. However, it has remained unclear how its transcriptional activation activity is regulated. Here, we show that the activated kinase activity of ERK5 is required for the C-terminal-half to enhance the AP-1 activity, and that the activated ERK5 undergoes autophosphorylation on its most C-terminal region. Changing these phosphorylatable threonine and serine residues to unphosphorylatable alanines significantly reduces the transcriptional activation activity of ERK5. Moreover, phosphomimetic mutants of the C-terminal-half of ERK5 without an N-terminal kinase domain are shown to be able to enhance the AP-1 activity in fibroblastic cells. These results reveal the role of the stimulus-induced ERK5 autophosphorylation in regulation of gene expression.
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Bregeon, Jeremy, Gervaise Loirand, Pierre Pacaud, and Malvyne Rolli-Derkinderen. "Angiotensin II induces RhoA activation through SHP2-dependent dephosphorylation of the RhoGAP p190A in vascular smooth muscle cells." American Journal of Physiology-Cell Physiology 297, no. 5 (November 2009): C1062—C1070. http://dx.doi.org/10.1152/ajpcell.00174.2009.
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Angiotensin II (ANG II) is a major regulator of blood pressure that essentially acts through activation of ANG II type 1 receptor (AT1R) of vascular smooth muscle cells (VSMC). AT1R activates numerous intracellular signaling pathways, including the small G protein RhoA known to control several VSMC functions. Nevertheless, the mechanisms leading to RhoA activation by AT1R are unknown. RhoA activation can result from activation of RhoA exchange factor and/or inhibition of Rho GTPase-activating protein (GAP). Here we hypothesize that a RhoGAP could participate to RhoA activation induced by ANG II in rat aortic VSMC. The knockdown of the RhoGAP p190A by small interfering RNA (siRNA) abolishes the activation of RhoA-Rho kinase pathway induced after 5 min of ANG II (0.1 μM) stimulation in rat aortic VSMC. We then show that AT1R activation induces p190A dephosphorylation and inactivation. In addition, expression of catalytically inactive or phosphoresistant p190A mutants increases the basal activity of RhoA-Rho kinase pathway, whereas phosphomimetic mutant inhibits early RhoA activation by ANG II. Using siRNA and mutant overexpression, we then demonstrate that the tyrosine phosphatase SHP2 is necessary for 1) maintaining p190A basally phosphorylated and activated by the tyrosine kinase c-Abl, and 2) inducing p190A dephosphorylation and RhoA activation in response to AT1R activation. Our work then defines p190A as a new mediator of RhoA activation by ANG II in VSMC.
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Feng, Ye, Wenjing Bao, Yanli Luo, Ling Tian, Xiafang Chen, Miaoying Yi, Hui Xiong, and Qian Huang. "Phosphomimetic Mutants of Pigment Epithelium-Derived Factor with Enhanced Anti-Choroidal Melanoma Cell Activity In Vitro and In Vivo." Investigative Opthalmology & Visual Science 53, no. 11 (October 3, 2012): 6793. http://dx.doi.org/10.1167/iovs.12-10326.
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Schlecht, William, Zhiqun Zhou, King-Lun Li, Daniel Rieck, Yexin Ouyang, and Wen-Ji Dong. "FRET study of the structural and kinetic effects of PKC phosphomimetic cardiac troponin T mutants on thin filament regulation." Archives of Biochemistry and Biophysics 550-551 (May 2014): 1–11. http://dx.doi.org/10.1016/j.abb.2014.03.013.
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Gianni, Davide, Nicolas Taulet, Céline DerMardirossian, and Gary M. Bokoch. "c-Src–Mediated Phosphorylation of NoxA1 and Tks4 Induces the Reactive Oxygen Species (ROS)–Dependent Formation of Functional Invadopodia in Human Colon Cancer Cells." Molecular Biology of the Cell 21, no. 23 (December 2010): 4287–98. http://dx.doi.org/10.1091/mbc.e10-08-0685.
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The NADPH oxidase family, consisting of Nox1-5 and Duox1-2, catalyzes the regulated formation of reactive oxygen species (ROS). Highly expressed in the colon, Nox1 needs the organizer subunit NoxO1 and the activator subunit NoxA1 for its activity. The tyrosine kinase c-Src is necessary for the formation of invadopodia, phosphotyrosine-rich structures which degrade the extracellular matrix (ECM). Many Src substrates are invadopodia components, including the novel Nox1 organizer Tks4 and Tks5 proteins. Nox1-dependent ROS generation is necessary for the maintenance of functional invadopodia in human colon cancer cells. However, the signals and the molecular machinery involved in the redox-dependent regulation of invadopodia formation remain unclear. Here, we show that the interaction of NoxA1 and Tks proteins is dependent on Src activity. Interestingly, the abolishment of Src-mediated phosphorylation of Tyr110 on NoxA1 and of Tyr508 on Tks4 blocks their binding and decreases Nox1-dependent ROS generation. The contemporary presence of Tks4 and NoxA1 unphosphorylable mutants blocks SrcYF-induced invadopodia formation and ECM degradation, while the overexpression of Tks4 and NoxA1 phosphomimetic mutants rescues this phenotype. Taken together, these results elucidate the role of c-Src activity on the formation of invadopodia and may provide insight into the mechanisms of tumor formation in colon cancers.
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Akai, Yuko, Ryuta Kanai, Norihiko Nakazawa, Masahiro Ebe, Chikashi Toyoshima, and Mitsuhiro Yanagida. "ATPase-dependent auto-phosphorylation of the open condensin hinge diminishes DNA binding." Open Biology 4, no. 12 (December 2014): 140193. http://dx.doi.org/10.1098/rsob.140193.
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Condensin, which contains two structural maintenance of chromosome (SMC) subunits and three regulatory non-SMC subunits, is essential for many chromosomal functions, including mitotic chromosome condensation and segregation. The ATPase domain of the SMC subunit comprises two termini connected by a long helical domain that is interrupted by a central hinge. The role of the ATPase domain has remained elusive. Here we report that the condensin SMC subunit of the fission yeast Schizosaccharomyces pombe is phosphorylated in a manner that requires the presence of the intact SMC ATPase Walker motif. Principal phosphorylation sites reside in the conserved, glycine-rich stretch at the hinge interface surrounded by the highly basic DNA-binding patch. Phosphorylation reduces affinity for DNA. Consistently, phosphomimetic mutants produce severe mitotic phenotypes. Structural evidence suggests that prior opening (though slight) of the hinge is necessary for phosphorylation, which is implicated in condensin's dissociation from and its progression along DNA.
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Routray, Chittaranjan, Chunsheng Liu, Usman Yaqoob, Daniel D. Billadeau, Kenneth D. Bloch, Kozo Kaibuchi, Vijay H. Shah, and Ningling Kang. "Protein kinase G signaling disrupts Rac1-dependent focal adhesion assembly in liver specific pericytes." American Journal of Physiology-Cell Physiology 301, no. 1 (July 2011): C66—C74. http://dx.doi.org/10.1152/ajpcell.00038.2011.
Full textAbstract:
Nitric oxide (NO) regulates the function of perivascular cells (pericytes), including hepatic stellate cells (HSC), mainly by activating cGMP and cGMP-dependent kinase (PKG) via NO/cGMP paracrine signaling. Although PKG is implicated in integrin-mediated cell adhesion to extracellular matrix, whether or how PKG signaling regulates the assembly of focal adhesion complexes (FA) and migration of HSC is not known. With the help of complementary molecular and cell biological approaches, we demonstrate here that activation of PKG signaling in HSC inhibits vascular tubulogenesis, migration/chemotaxis, and assembly of mature FA plaques, as assessed by vascular tubulogenesis assays and immunofluorescence localization of FA markers such as vinculin and vasodilator-stimulated phosphoprotein (VASP). To determine whether PKG inhibits FA assembly by phosphorylation of VASP at Ser-157, Ser-239, and Thr-278, we mutated these putative phosphorylation sites to alanine (VASP3A, phosphoresistant mutant) or aspartic acid (VASP3D, phosphomimetic), respectively. Data generated from these two mutants suggest that the effect of PKG on FA is independent of these three phosphorylation sites. In contrast, activation of PKG inhibits the activity of small GTPase Rac1 and its association with the effector protein IQGAP1. Moreover, PKG activation inhibits the formation of a trimeric protein complex containing Rac1, IQGAP1, and VASP. Finally, we found that expression of a constitutively active Rac1 mutant abolishes the inhibitory effects of PKG on FA formation. In summary, our data suggest that activation of PKG signaling in pericytes inhibits FA formation by inhibiting Rac1.
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Chen, Cailin, François Agnès, and Céline Gélinas. "Mapping of a Serine-Rich Domain Essential for the Transcriptional, Antiapoptotic, and Transforming Activities of the v-Rel Oncoprotein." Molecular and Cellular Biology 19, no. 1 (January 1, 1999): 307–16. http://dx.doi.org/10.1128/mcb.19.1.307.
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ABSTRACT The v-Rel oncoprotein belongs to the Rel/NF-κB family of transcription factors and induces aggressive lymphomas in chickens and transgenic mice. Current models for cell transformation by v-Rel invoke the combined activation of gene expression and the dominant inhibition of transcription mediated by its cellular homologs. Here, we mapped a serine-rich transactivation domain in the C terminus of v-Rel that is necessary for its biological activity. Specific serine-to-alanine substitutions within this region impaired the transcriptional activity of v-Rel, whereas a double mutant abolished its function. In contrast, substitutions with phosphomimetic aspartate residues led to a complete recovery of the transcriptional potential. The transforming activity of v-Rel mutants correlated with their ability to inhibit programmed cell death. The transforming and antiapoptotic activities of v-Rel were abolished by defined Ser-to-Ala mutations and restored by most Ser-to-Asp substitutions. However, one Ser-to-Asp mutant showed wild-type transactivation ability but failed to block apoptosis and to transform cells. These results show that the transactivation function of v-Rel is necessary but not sufficient for cell transformation, adding an important dimension to the transformation model. It is possible that defined protein-protein interactions are also required to block apoptosis and transform cells. Since v-Rel is an acutely oncogenic member of the Rel/NF-κB family, our data raise the possibility that phosphorylation of its serine-rich transactivation domain may regulate its unique biological activity.
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Lyons, Patrick D., Grantley R. Peck, Arminja N. Kettenbach, Scott A. Gerber, Liya Roudaia, and Gustav E. Lienhard. "Insulin stimulates the phosphorylation of the exocyst protein Sec8 in adipocytes." Bioscience Reports 29, no. 4 (May 7, 2009): 229–35. http://dx.doi.org/10.1042/bsr20080162.
Full textAbstract:
The signal transduction pathway leading from the insulin receptor to stimulate the fusion of vesicles containing the glucose transporter GLUT4 with the plasma membrane in adipocytes and muscle cells is not completely understood. Current evidence suggests that in addition to the Rab GTPase-activating protein AS160, at least one other substrate of Akt (also called protein kinase B), which is as yet unidentified, is required. Sec8 is a component of the exocyst complex that has been previously implicated in GLUT4 trafficking. In the present study, we report that insulin stimulates the phosphorylation of Sec8 on Ser-32 in 3T3-L1 adipocytes. On the basis of the sequence around Ser-32 and the finding that phosphorylation is inhibited by the PI3K (phosphoinositide 3-kinase) inhibitor wortmannin, it is likely that Akt is the kinase for Ser-32. We examined the possible role of Ser-32 phosphorylation in the insulin-stimulated trafficking of GLUT4, as well as the TfR (transferrin receptor), to the plasma membrane by determining the effects of overexpression of the non-phosphorylatable S32A mutant of Sec8 and the phosphomimetic S32E mutant of Sec8. Substantial overexpression of both mutants had no effect on the amount of GLUT4 or TfR at the cell surface in either the untreated or insulin-treated states. These results indicate that insulin-stimulated phosphorylation of Sec8 is not part of the mechanism by which insulin enhances the fusion of vesicles with the plasma membrane.
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Zaytsev, Anatoly V., Lynsie J. R. Sundin, Keith F. DeLuca, Ekaterina L. Grishchuk, and Jennifer G. DeLuca. "Accurate phosphoregulation of kinetochore–microtubule affinity requires unconstrained molecular interactions." Journal of Cell Biology 206, no. 1 (June 30, 2014): 45–59. http://dx.doi.org/10.1083/jcb.201312107.
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Accurate chromosome segregation relies on dynamic interactions between microtubules (MTs) and the NDC80 complex, a major kinetochore MT-binding component. Phosphorylation at multiple residues of its Hec1 subunit may tune kinetochore–MT binding affinity for diverse mitotic functions, but molecular details of such phosphoregulation remain elusive. Using quantitative analyses of mitotic progression in mammalian cells, we show that Hec1 phosphorylation provides graded control of kinetochore–MT affinity. In contrast, modeling the kinetochore interface with repetitive MT binding sites predicts a switchlike response. To reconcile these findings, we hypothesize that interactions between NDC80 complexes and MTs are not constrained, i.e., the NDC80 complexes can alternate their binding between adjacent kinetochore MTs. Experiments using cells with phosphomimetic Hec1 mutants corroborate predictions of such a model but not of the repetitive sites model. We propose that accurate regulation of kinetochore–MT affinity is driven by incremental phosphorylation of an NDC80 molecular “lawn,” in which the NDC80–MT bonds reorganize dynamically in response to the number and stability of MT attachments.
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Aratyn, Yvonne S., Thomas E. Schaus, Edwin W. Taylor, and Gary G. Borisy. "Intrinsic Dynamic Behavior of Fascin in Filopodia." Molecular Biology of the Cell 18, no. 10 (October 2007): 3928–40. http://dx.doi.org/10.1091/mbc.e07-04-0346.
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Recent studies showed that the actin cross-linking protein, fascin, undergoes rapid cycling between filopodial filaments. Here, we used an experimental and computational approach to dissect features of fascin exchange and incorporation in filopodia. Using expression of phosphomimetic fascin mutants, we determined that fascin in the phosphorylated state is primarily freely diffusing, whereas actin bundling in filopodia is accomplished by fascin dephosphorylated at serine 39. Fluorescence recovery after photobleaching analysis revealed that fascin rapidly dissociates from filopodial filaments with a kinetic off-rate of 0.12 s−1 and that it undergoes diffusion at moderate rates with a coefficient of 6 μm2s−1. This kinetic off-rate was recapitulated in vitro, indicating that dynamic behavior is intrinsic to the fascin cross-linker. A computational reaction–diffusion model showed that reversible cross-linking is required for the delivery of fascin to growing filopodial tips at sufficient rates. Analysis of fascin bundling indicated that filopodia are semiordered bundles with one bound fascin per 25–60 actin monomers.
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Gandy, K. Alexa Orr, Daniel Canals, Mohamad Adada, Masayuki Wada, Patrick Roddy, Ashley J. Snider, Yusuf A. Hannun, and Lina M. Obeid. "Sphingosine 1-phosphate induces filopodia formation through S1PR2 activation of ERM proteins." Biochemical Journal 449, no. 3 (January 9, 2013): 661–72. http://dx.doi.org/10.1042/bj20120213.
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Previously we demonstrated that the sphingolipids ceramide and S1P (sphingosine 1-phosphate) regulate phosphorylation of the ERM (ezrin/radixin/moesin) family of cytoskeletal proteins [Canals, Jenkins, Roddy, Hernande-Corbacho, Obeid and Hannun (2010) J. Biol. Chem. 285, 32476–3285]. In the present article, we show that exogenously applied or endogenously generated S1P (in a sphingosine kinase-dependent manner) results in significant increases in phosphorylation of ERM proteins as well as filopodia formation. Using phosphomimetic and non-phosphorylatable ezrin mutants, we show that the S1P-induced cytoskeletal protrusions are dependent on ERM phosphorylation. Employing various pharmacological S1PR (S1P receptor) agonists and antagonists, along with siRNA (small interfering RNA) techniques and genetic knockout approaches, we identify the S1PR2 as the specific and necessary receptor to induce phosphorylation of ERM proteins and subsequent filopodia formation. Taken together, the results demonstrate a novel mechanism by which S1P regulates cellular architecture that requires S1PR2 and subsequent phosphorylation of ERM proteins.
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Chan, Ying Wai, A. Arockia Jeyaprakash, Erich A. Nigg, and Anna Santamaria. "Aurora B controls kinetochore–microtubule attachments by inhibiting Ska complex–KMN network interaction." Journal of Cell Biology 196, no. 5 (February 27, 2012): 563–71. http://dx.doi.org/10.1083/jcb.201109001.
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The KMN network (named according to the acronym for KNL1, Mis12, and Ndc80) and the more recently identified Ska complex (Ska1–3) have been shown to mediate kinetochore (KT)–microtubule (MT) attachments. How these two complexes cooperate to achieve stable end-on attachments remains unknown. In this paper, we show that Aurora B negatively regulates the localization of the Ska complex to KTs and that recruitment of the Ska complex to KTs depends on the KMN network. We identified interactions between members of the KMN and Ska complexes and demonstrated that these interactions are regulated by Aurora B. Aurora B directly phosphorylated Ska1 and Ska3 in vitro, and expression of phosphomimetic mutants of Ska1 and Ska3 impaired Ska KT recruitment and formation of stable KT–MT fibers (K-fibers), disrupting mitotic progression. We propose that Aurora B phosphorylation antagonizes the interaction between the Ska complex and the KMN network, thereby controlling Ska recruitment to KTs and stabilization of KT–MT attachments.
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Meng, Fanrui, Sandeep Saxena, Youtao Liu, Bharat Joshi, Timothy H. Wong, Jay Shankar, Leonard J. Foster, Pascal Bernatchez, and Ivan R. Nabi. "The phospho–caveolin-1 scaffolding domain dampens force fluctuations in focal adhesions and promotes cancer cell migration." Molecular Biology of the Cell 28, no. 16 (August 2017): 2190–201. http://dx.doi.org/10.1091/mbc.e17-05-0278.
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Caveolin-1 (Cav1), a major Src kinase substrate phosphorylated on tyrosine-14 (Y14), contains the highly conserved membrane-proximal caveolin scaffolding domain (CSD; amino acids 82–101). Here we show, using CSD mutants (F92A/V94A) and membrane-permeable CSD-competing peptides, that Src kinase–dependent pY14Cav1 regulation of focal adhesion protein stabilization, focal adhesion tension, and cancer cell migration is CSD dependent. Quantitative proteomic analysis of Cav1-GST (amino acids 1–101) pull downs showed sixfold-increased binding of vinculin and, to a lesser extent, α-actinin, talin, and filamin, to phosphomimetic Cav1Y14D relative to nonphosphorylatable Cav1Y14F. Consistently, pY14Cav1 enhanced CSD-dependent vinculin tension in focal adhesions, dampening force fluctuation and synchronously stabilizing cellular focal adhesions in a high-tension mode, paralleling effects of actin stabilization. This identifies pY14Cav1 as a molecular regulator of focal adhesion tension and suggests that functional interaction between Cav1 Y14 phosphorylation and the CSD promotes focal adhesion traction and, thereby, cancer cell motility.
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Boese, Cody J., Jonathan Nye, Daniel W. Buster, Tiffany A. McLamarrah, Amy E. Byrnes, Kevin C. Slep, Nasser M. Rusan, and Gregory C. Rogers. "Asterless is a Polo-like kinase 4 substrate that both activates and inhibits kinase activity depending on its phosphorylation state." Molecular Biology of the Cell 29, no. 23 (November 15, 2018): 2874–86. http://dx.doi.org/10.1091/mbc.e18-07-0445.
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Centriole assembly initiates when Polo-like kinase 4 (Plk4) interacts with a centriole “targeting-factor.” In Drosophila, Asterless/Asl (Cep152 in humans) fulfills the targeting role. Interestingly, Asl also regulates Plk4 levels. The N-terminus of Asl (Asl-A; amino acids 1-374) binds Plk4 and promotes Plk4 self-destruction, although it is unclear how this is achieved. Moreover, Plk4 phosphorylates the Cep152 N-terminus, but the functional consequence is unknown. Here, we show that Plk4 phosphorylates Asl and mapped 13 phospho-residues in Asl-A. Nonphosphorylatable alanine (13A) and phosphomimetic (13PM) mutants did not alter Asl function, presumably because of the dominant role of the Asl C-terminus in Plk4 stabilization and centriolar targeting. To address how Asl-A phosphorylation specifically affects Plk4 regulation, we generated Asl-A fragment phospho-mutants and expressed them in cultured Drosophila cells. Asl-A-13A stimulated kinase activity by relieving Plk4 autoinhibition. In contrast, Asl-A-13PM inhibited Plk4 activity by a novel mechanism involving autophosphorylation of Plk4’s kinase domain. Thus, Asl-A’s phosphorylation state determines which of Asl-A’s two opposing effects are exerted on Plk4. Initially, nonphosphorylated Asl binds Plk4 and stimulates its kinase activity, but after Asl is phosphorylated, a negative-feedback mechanism suppresses Plk4 activity. This dual regulatory effect by Asl-A may limit Plk4 to bursts of activity that modulate centriole duplication.
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Gangwal, Aakriti, Nitika Sangwan, Neha Dhasmana, Nishant Kumar, Chetkar Chandra Keshavam, Lalit K. Singh, Ankur Bothra, et al. "Role of serine/threonine protein phosphatase PrpN in the life cycle of Bacillus anthracis." PLOS Pathogens 18, no. 8 (August 1, 2022): e1010729. http://dx.doi.org/10.1371/journal.ppat.1010729.
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Reversible protein phosphorylation at serine/threonine residues is one of the most common protein modifications, widely observed in all kingdoms of life. The catalysts controlling this modification are specific serine/threonine kinases and phosphatases that modulate various cellular pathways ranging from growth to cellular death. Genome sequencing and various omics studies have led to the identification of numerous serine/threonine kinases and cognate phosphatases, yet the physiological relevance of many of these proteins remain enigmatic. In Bacillus anthracis, only one ser/thr phosphatase, PrpC, has been functionally characterized; it was reported to be non-essential for bacterial growth and survival. In the present study, we characterized another ser/thr phosphatase (PrpN) of B. anthracis by various structural and functional approaches. To examine its physiological relevance in B. anthracis, a null mutant strain of prpN was generated and shown to have defects in sporulation and reduced synthesis of toxins (PA and LF) and the toxin activator protein AtxA. We also identified CodY, a global transcriptional regulator, as a target of PrpN and ser/thr kinase PrkC. CodY phosphorylation strongly controlled its binding to the promoter region of atxA, as shown using phosphomimetic and phosphoablative mutants. In nutshell, the present study reports phosphorylation-mediated regulation of CodY activity in the context of anthrax toxin synthesis in B. anthracis by a previously uncharacterized ser/thr protein phosphatase–PrpN.