The hydrophobic motif of ROCK2 requires association with the N-terminal extension for kinase activity

2009 ◽  
Vol 419 (1) ◽  
pp. 141-148 ◽  
Author(s):  
Amber L. Couzens ◽  
Vivian Saridakis ◽  
Michael P. Scheid
Keyword(s):  
Growth Factor ◽  
Kinase Activity ◽  
Coiled Coil ◽  
Kinase Domain ◽  
Agc Kinases ◽  
Threonine Residue ◽  
Substrate Phosphorylation ◽  
Hydrophobic Motif

ROCK (Rho-associated coiled-coil kinase) 2 is a member of the AGC kinase family that plays an essential role downstream of Rho in actin cytoskeleton assembly and contractility. The process of ROCK2 activation is complex and requires suppression of an autoinhibitory mechanism that is facilitated by Rho binding. ROCK2 harbours a C-terminal extension within the kinase domain that contains a hydrophobic cluster of phenylalanine and tyrosine residues surrounding a key threonine residue. In growth-factor-stimulated AGC kinases, the hydrophobic motif is important for the transition of the kinase from inactive to active complex and requires phosphorylation of the conserved serine/threonine residue. Less is understood about the contribution that the hydrophobic motif plays in the activation of ROCK, and the role of the hydrophobic motif threonine at position 405. In the present study, we show that this residue of ROCK is essential for substrate phosphorylation and kinase domain dimerization. However, in contrast with the growth-factor-activated AGC kinases, a phosphomimetic residue at position 405 was inhibitory for ROCK2 activity and dimerization. A soluble hydrophobic motif peptide allosterically activated ROCK2 In vitro, but not the equivalent peptide with Asp405 substitution. Mechanistically, both ROCK2 activity and dimerization were dependent upon the interaction between Thr405 of the hydrophobic motif and Asp39 of the N-terminal extension. The reciprocal exchange of these residues was permissive for kinase activity, but dimerization was lost. These results support the rationale for development of small-molecule inhibitors designed to block ROCK activation by selectively interfering with hydrophobic motif-mediated activation-state transition and dimer formation.

scholarly journals Mapping of a self-interaction domain of the cytomegalovirus protein kinase pUL97

2007 ◽  
Vol 88 (2) ◽  
pp. 395-404 ◽  
Author(s):  
Vera Schregel ◽  
Sabrina Auerochs ◽  
Ramona Jochmann ◽  
Katja Maurer ◽  
Thomas Stamminger ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Protein Kinase Activity ◽  
Interaction Domain ◽  
Amino Acid Region ◽  
Substrate Phosphorylation ◽  
Regulatory Functions ◽  
Acid Region

The human cytomegalovirus-encoded protein kinase pUL97 is a determinant of efficient virus replication and fulfils several regulatory functions. In particular, pUL97 interacts with and phosphorylates viral and cellular proteins. Substrate phosphorylation has regulatory consequences on viral replicative stages such as DNA synthesis, transcription and nuclear capsid egress. pUL97, in accordance with related herpesviral protein kinases, possesses strong autophosphorylation activity. Here, we demonstrate that pUL97 shows a pronounced potential to self-interact. Self-interaction of pUL97 is not dependent on its kinase activity, as seen with a catalytically inactive point mutant. The property of self-interaction maps to the amino acid region 231–280 which is separable from the postulated kinase domain. The detection of high-molecular-mass complexes of pUL97 suggests the formation of dimers and oligomers. Importantly, the analysis of pUL97 mutants by in vitro kinase assays demonstrated a correlation between self-interaction and protein kinase activity, i.e. all mutants lacking the ability to self-interact were negative or reduced in their kinase activity. Thus, our findings provide novel insights into the pUL97 structure–activity relationship suggesting an importance of self-interaction for pUL97 functionality.

scholarly journals Antibodies to insulin receptor tyrosine kinase stimulate its activity towards exogenous substrates without inducing receptor autophosphorylation

1989 ◽  
Vol 260 (3) ◽  
pp. 749-756 ◽  
Author(s):  
V Baron ◽  
N Gautier ◽  
N Rochet ◽  
R Ballotti ◽  
B Rossi ◽  
...  
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Insulin Receptor ◽  
Receptor Tyrosine Kinase ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Insulin Receptor Tyrosine Kinase ◽  
Substrate Phosphorylation ◽  
Exogenous Substrates

Anti-peptide antibodies directed against a highly-conserved sequence of the insulin receptor tyrosine kinase domain have been used to study the relationship between this specific region and kinase activation. Antibodies have been prepared by the injection into a rabbit of a synthetic peptide (P2) corresponding to residues 1110-1125 of the proreceptor. The peptide exhibits 88-95% sequence similarity with the corresponding sequence in the v-ros protein and in receptors for epidermal growth factor and for insulin-like growth factor 1. Two antibodies with different specificities could be separated from total antiserum obtained after immunization with P2. One antibody [anti-(P-Tyr)] cross-reacted with phosphotyrosine and immunoprecipitated solely autophosphorylated receptors. This antibody was shown to increase or decrease the receptor tyrosine kinase activity depending on its concentration. In all circumstances receptor autophosphorylation and substrate phosphorylation were modulated in a parallel fashion. The second antibody (anti-P2) failed to immunoprecipitate the insulin receptor, but was found to interact with both the peptide and the receptor by e.l.i.s.a. assay. Using a tyrosine co-polymer we found that anti-P2 activated the insulin receptor kinase leading to substrate phosphorylation at a level similar to that observed with insulin. This effect was additive to the hormonal effect. In contrast, receptor autophosphorylation was not modified by the anti-peptide. The differential effect of this anti-peptide further supports the idea that receptor autophosphorylation and kinase activity towards exogenous substrates might be independently regulated. Finally, our data suggest that conformational changes in the receptor tyrosine kinase domain may be sufficient for activation of its enzymic activity.

scholarly journals Characterization of the kinase activity of a WNK4 protein complex

2009 ◽  
Vol 297 (3) ◽  
pp. F685-F692 ◽  
Author(s):  
Robert Ahlstrom ◽  
Alan S. L. Yu
Keyword(s):  
Kinase Activity ◽  
Genetic Disorder ◽  
Signal Transduction Pathway ◽  
Kinase Domain ◽  
Full Length ◽  
Hek293 Cells ◽  
Pseudohypoaldosteronism Type Ii

Mutations in WNK4 protein kinase cause pseudohypoaldosteronism type II (PHAII), a genetic disorder that is characterized by renal NaCl and K+ retention leading to hypertension and hyperkalemia. Consistent with this, WNK4 is known to regulate several renal tubule transporters, including the NaCl cotransporter, NCC, and the K+ channel, ROMK, but the mechanisms are incompletely understood, and the role of the kinase activity in its actions is highly controversial. To assay WNK4 kinase activity, we have now succeeded in expressing and purifying full-length, enzymatically active WNK4 protein from HEK293 cells. We show that full-length wild-type WNK4 phosphorylates oxidative stress response kinase 1 (OSR1) and Ste20/SPS1-related proline/alanine-rich kinase (SPAK) in vitro. Introducing the PHAII-associated mutations, E559K, D561A, and Q562E, into our protein had no significant effect on this phosphorylation. We conclude that PHAII is unlikely to be caused by abnormal WNK4 kinase activity. We also made the intriguing observation that inactivating mutations of the WNK4 kinase domain did not completely abolish in vitro phosphorylation of OSR1/SPAK. Led by this, we identified a novel 40-kDa kinase that associates specifically with the COOH-terminal half of WNK4 and is able to phosphorylate both WNK4 and SPAK/OSR1. We suggest that this 40-kDa kinase functions in the WNK4 signal transduction pathway and may mediate some of the physiological actions attributed to WNK4.

scholarly journals Autoinhibition of the Platelet-derived Growth Factor β-Receptor Tyrosine Kinase by Its C-terminal Tail

2004 ◽  
Vol 279 (19) ◽  
pp. 19732-19738 ◽  
Author(s):  
Federica Chiara ◽  
Subal Bishayee ◽  
Carl-Henrik Heldin ◽  
Jean-Baptiste Demoulin
Keyword(s):  
Growth Factor ◽  
Kinase Activity ◽  
Platelet Derived Growth Factor ◽  
Receptor Kinase ◽  
Wild Type ◽  
Focus Formation ◽  
Soluble Peptide ◽  
Β Receptor

In this report, we investigated the role of the C-terminal tail of the platelet-derived growth factor (PDGF) β-receptor in the control of the receptor kinase activity. Using a panel of PDGF β-receptor mutants with progressive C-terminal truncations, we observed that deletion of the last 46 residues, which contain a proline- and glutamic acid-rich motif, increased the autoactivation velocityin vitroand theVmaxof the phosphotransfer reaction, in the absence of ligand, as compared with wild-type receptors. By contrast, the kinase activity of mutant and wild-type receptors that were pre-activated by treatment with PDGF was comparable. Using a conformation-sensitive antibody, we found that truncated receptors presented an active conformation even in the absence of PDGF. A soluble peptide containing the Pro/Glu-rich motif specifically inhibited the PDGF β-receptor kinase activity. Whereas deletion of this motif was not enough to confer ligand-independent transforming ability to the receptor, it dramatically enhanced the effect of the weakly activating D850N mutation in a focus formation assay. These findings indicate that allosteric inhibition of the PDGF β-receptor by its C-terminal tail is one of the mechanisms involved in keeping the receptor inactive in the absence of ligand.

scholarly journals mTORC2 controls the activity of PKC and Akt by phosphorylating a conserved TOR interaction motif

2021 ◽  
Vol 14 (678) ◽  
pp. eabe4509
Author(s):  
Timothy R. Baffi ◽  
Gema Lordén ◽  
Jacob M. Wozniak ◽  
Andreas Feichtner ◽  
Wayland Yeung ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Activation Loop ◽  
Dimer Interface ◽  
Agc Kinases ◽  
Evolutionarily Conserved ◽  
Phosphorylation Event ◽  
Hydrophobic Motif

The complex mTORC2 is accepted to be the kinase that controls the phosphorylation of the hydrophobic motif, a key regulatory switch for AGC kinases, although whether mTOR directly phosphorylates this motif remains controversial. Here, we identified an mTOR-mediated phosphorylation site that we termed the TOR interaction motif (TIM; F-x3-F-pT), which controls the phosphorylation of the hydrophobic motif of PKC and Akt and the activity of these kinases. The TIM is invariant in mTORC2-dependent AGC kinases, is evolutionarily conserved, and coevolved with mTORC2 components. Mutation of this motif in Akt1 and PKCβII abolished cellular kinase activity by impairing activation loop and hydrophobic motif phosphorylation. mTORC2 directly phosphorylated the PKC TIM in vitro, and this phosphorylation event was detected in mouse brain. Overexpression of PDK1 in mTORC2-deficient cells rescued hydrophobic motif phosphorylation of PKC and Akt by a mechanism dependent on their intrinsic catalytic activity, revealing that mTORC2 facilitates the PDK1 phosphorylation step, which, in turn, enables autophosphorylation. Structural analysis revealed that PKC homodimerization is driven by a TIM-containing helix, and biophysical proximity assays showed that newly synthesized, unphosphorylated PKC dimerizes in cells. Furthermore, disruption of the dimer interface by stapled peptides promoted hydrophobic motif phosphorylation. Our data support a model in which mTORC2 relieves nascent PKC dimerization through TIM phosphorylation, recruiting PDK1 to phosphorylate the activation loop and triggering intramolecular hydrophobic motif autophosphorylation. Identification of TIM phosphorylation and its role in the regulation of PKC provides the basis for AGC kinase regulation by mTORC2.

scholarly journals An ordered pattern of Ana2 phosphorylation by Plk4 is required for centriole assembly

2017 ◽  
Author(s):  
Tiffany A. McLamarrah ◽  
Daniel W. Buster ◽  
Brian J. Galletta ◽  
Cody J. Boese ◽  
John M. Ryniawec ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Coiled Coil ◽  
Kinase Domain ◽  
Early Step ◽  
Centriole Duplication ◽  
Physiological Relevance ◽  
Phosphorylation Pattern ◽  
Centriole Assembly ◽  
Assembly Site

AbstractPolo-like kinase 4 (Plk4) initiates an early step in centriole assembly by phosphorylating Ana2/STIL, a structural component of the procentriole. Here, we show that Plk4 binding to the central coiled-coil (CC) of Ana2 is a conserved event, involving Polo-box 3 and a previously unidentified putative CC located adjacent to the kinase domain. Ana2 binding stimulates Plk4 kinase activity in vitro, and, in turn, is phosphorylated along its length. Previous studies showed that Plk4 phosphorylates the C-terminal STAN domain of Ana2/STIL, triggering binding and recruitment of the cartwheel protein Sas6 to the procentriole assembly site. However, the physiological relevance of N-terminal phosphorylation was unknown. We found that Plk4 first phosphorylates the extreme N–terminus of Ana2 which is critical for subsequent STAN domain modification. Phosphorylation of the central region then breaks the Plk4-Ana2 interaction. This phosphorylation pattern is important for centriole assembly and integrity because replacement of endogenous Ana2 with phospho-Ana2 mutants disrupts distinct steps in Ana2 function and inhibits centriole duplication.

Activation of bovine oocytes by protein synthesis inhibitors: new findings on the role of MPF/MAPKs†

2021 ◽  
Author(s):  
Cecilia Valencia ◽  
Felipe Alonso Pérez ◽  
Carola Matus ◽  
Ricardo Felmer ◽  
María Elena Arias
Keyword(s):  
Protein Synthesis ◽  
Kinase Activity ◽  
Cyclin B1 ◽  
Protein Synthesis Inhibitors ◽  
Vitro Fertilization ◽  
New Findings ◽  
Bovine Oocytes ◽  
Dependent Pathway

Abstract The present study evaluated the mechanism by which protein synthesis inhibitors activate bovine oocytes. The aim was to analyze the dynamics of MPF and MAPKs. MII oocytes were activated with ionomycin (Io), ionomycin+anisomycin (ANY) and ionomycin+cycloheximide (CHX) and by in vitro fertilization (IVF). The expression of cyclin B1, p-CDK1, p-ERK1/2, p-JNK, and p-P38 were evaluated by immunodetection and the kinase activity of ERK1/2 was measured by enzyme assay. Evaluations at 1, 4, and 15 hours postactivation (hpa) showed that the expression of cyclin B1 was not modified by the treatments. ANY inactivated MPF by p-CDK1Thr14-Tyr15 at 4 hpa (P < 0.05), CHX increased pre-MPF (p-CDK1Thr161 and p-CDK1Thr14-Tyr15) at 1 hpa and IVF increased p-CDK1Thr14-Tyr15 at 17 hours postfertilization (hpf) (P < 0.05). ANY and CHX reduced the levels of p-ERK1/2 at 4 hpa (P < 0.05) and its activity at 4 and 1 hpa, respectively (P < 0.05). Meanwhile, IVF increased p-ERK1/2 at 6 hpf (P < 0.05); however, its kinase activity decreased at 6 hpf (P < 0.05). p-JNK in ANY, CHX, and IVF oocytes decreased at 4 hpa (P < 0.05). p-P38 was only observed at 1 hpa, with no differences between treatments. In conclusion, activation of bovine oocytes by ANY, CHX, and IVF inactivates MPF by CDK1-dependent specific phosphorylation without cyclin B1 degradation. ANY or CHX promoted this inactivation, which seemed to be more delayed in the physiological activation (IVF). Both inhibitors modulated MPF activity via an ERK1/2-independent pathway, whereas IVF activated the bovine oocytes via an ERK1/2-dependent pathway. Finally, ANY does not activate the JNK and P38 kinase pathways.

scholarly journals Hyperglycaemia up-regulates placental growth factor (PlGF) expression and secretion in endothelial cells via suppression of PI3 kinase-Akt signalling and activation of FOXO1

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Samir Sissaoui ◽  
Stuart Egginton ◽  
Ling Ting ◽  
Asif Ahmed ◽  
Peter W. Hewett
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Endothelial Dysfunction ◽  
Akt Activation ◽  
Forkhead Box ◽  
Pi3k Activity ◽  
Binding Sequence

AbstractPlacenta growth factor (PlGF) is a pro-inflammatory angiogenic mediator that promotes many pathologies including diabetic complications and atherosclerosis. Widespread endothelial dysfunction precedes the onset of these conditions. As very little is known of the mechanism(s) controlling PlGF expression in pathology we investigated the role of hyperglycaemia in the regulation of PlGF production in endothelial cells. Hyperglycaemia stimulated PlGF secretion in cultured primary endothelial cells, which was suppressed by IGF-1-mediated PI3K/Akt activation. Inhibition of PI3K activity resulted in significant PlGF mRNA up-regulation and protein secretion. Similarly, loss or inhibition of Akt activity significantly increased basal PlGF expression and prevented any further PlGF secretion in hyperglycaemia. Conversely, constitutive Akt activation blocked PlGF secretion irrespective of upstream PI3K activity demonstrating that Akt is a central regulator of PlGF expression. Knock-down of the Forkhead box O-1 (FOXO1) transcription factor, which is negatively regulated by Akt, suppressed both basal and hyperglycaemia-induced PlGF secretion, whilst FOXO1 gain-of-function up-regulated PlGF in vitro and in vivo. FOXO1 association to a FOXO binding sequence identified in the PlGF promoter also increased in hyperglycaemia. This study identifies the PI3K/Akt/FOXO1 signalling axis as a key regulator of PlGF expression and unifying pathway by which PlGF may contribute to common disorders characterised by endothelial dysfunction, providing a target for therapy.

scholarly journals Activity of the Ste20-like kinase, SLK, is enhanced by homodimerization

2011 ◽  
Vol 301 (3) ◽  
pp. F554-F564 ◽  
Author(s):  
Sierra Delarosa ◽  
Julie Guillemette ◽  
Joan Papillon ◽  
Ying-Shan Han ◽  
Arnold S. Kristof ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Gel Filtration ◽  
Coiled Coil ◽  
Kinase Domain ◽  
Kinase Assay ◽  
Luciferase Reporter ◽  
Fk506 Binding Protein ◽  
Induced Apoptosis ◽  
Injury And Repair

The expression and activation of the Ste20-like kinase, SLK, is increased during renal development and recovery from ischemic acute renal failure. SLK promotes apoptosis, and during renal injury and repair, transcriptional induction or posttranscriptional control of SLK may, therefore, regulate cell survival. SLK contains protein interaction (coiled-coil) domains, suggesting that posttranslational homodimerization may also modulate SLK activity. We therefore expressed coiled-coil regions in the C-terminal domain of SLK as fusion proteins and demonstrated their homodimerization. By gel-filtration chromatography, endogenous and heterologously expressed SLK were detected in a macromolecular protein complex. To test the role of homodimerization in kinase activation, we constructed a fusion protein consisting of the SLK catalytic domain (amino acids 1–373) and a modified FK506 binding protein, Fv (Fv-SLK 1–373). Addition of AP20187 (an analog of FK506) enhanced the homodimerization of Fv-SLK 1–373. In an in vitro kinase assay, the dimeric Fv-SLK 1–373 displayed greater kinase activity than the monomeric form. In cells expressing Fv-SLK 1–373, homodimerization increased activation-specific phosphorylation of the proapoptotic kinases, c-Jun N-terminal kinase and p38 kinase. Compared with the monomer, dimeric Fv-SLK 1–373 enhanced the activation of a Bax promoter-luciferase reporter. Finally, expression of Fv-SLK 1–373 induced apoptosis, and the effect was increased by homodimerization. Thus the activity, downstream signaling, and functional effects of SLK are enhanced by dimerization of the kinase domain.

scholarly journals Regulation of Jak2 Function by Phosphorylation of Tyr317 and Tyr637 during Cytokine Signaling

2009 ◽  
Vol 29 (12) ◽  
pp. 3367-3378 ◽  
Author(s):  
Scott A. Robertson ◽  
Rositsa I. Koleva ◽  
Lawrence S. Argetsinger ◽  
Christin Carter-Su ◽  
Jarrod A. Marto ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Feedback Inhibition ◽  
Dominant Role ◽  
Kinase Domain ◽  
Cytokine Signaling ◽  
Tyrosine Kinase Domain ◽  
Phosphorylation Sites ◽  
Cytokine Stimulation

ABSTRACT Jak2, the cognate tyrosine kinase for numerous cytokine receptors, undergoes multisite phosphorylation during cytokine stimulation. To understand the role of phosphorylation in Jak2 regulation, we used mass spectrometry to identify numerous Jak2 phosphorylation sites and characterize their significance for Jak2 function. Two sites outside of the tyrosine kinase domain, Tyr317 in the FERM domain and Tyr637 in the JH2 domain, exhibited strong regulation of Jak2 activity. Mutation of Tyr317 promotes increased Jak2 activity, and the phosphorylation of Tyr317 during cytokine signaling requires prior activation loop phosphorylation, which is consistent with a role for Tyr317 in the feedback inhibition of Jak2 kinase activity after receptor stimulation. Comparison to several previously identified regulatory phosphorylation sites on Jak2 revealed a dominant role for Tyr317 in the attenuation of Jak2 signaling. In contrast, mutation of Tyr637 decreased Jak2 signaling and activity and partially suppressed the activating JH2 V617F mutation, suggesting a role for Tyr637 phosphorylation in the release of JH2 domain-mediated suppression of Jak2 kinase activity during cytokine stimulation. The phosphorylation of Tyr317 and Tyr637 act in concert with other regulatory events to maintain appropriate control of Jak2 activity and cytokine signaling.

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