scholarly journals Kinase-interacting substrate screening is a novel method to identify kinase substrates

2015 ◽  
Vol 209 (6) ◽  
pp. 895-912 ◽  
Author(s):  
Mutsuki Amano ◽  
Tomonari Hamaguchi ◽  
Md. Hasanuzzaman Shohag ◽  
Kei Kozawa ◽  
Katsuhiro Kato ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Ternary Complex ◽  
Rho Kinase ◽  
Phosphorylation Sites ◽  
Dependent Manner ◽  
Cellular Functions ◽  
Associated Proteins ◽  
Novel Method ◽  
Kinase Substrates

Protein kinases play pivotal roles in numerous cellular functions; however, the specific substrates of each protein kinase have not been fully elucidated. We have developed a novel method called kinase-interacting substrate screening (KISS). Using this method, 356 phosphorylation sites of 140 proteins were identified as candidate substrates for Rho-associated kinase (Rho-kinase/ROCK2), including known substrates. The KISS method was also applied to additional kinases, including PKA, MAPK1, CDK5, CaMK1, PAK7, PKN, LYN, and FYN, and a lot of candidate substrates and their phosphorylation sites were determined, most of which have not been reported previously. Among the candidate substrates for Rho-kinase, several functional clusters were identified, including the polarity-associated proteins, such as Scrib. We found that Scrib plays a crucial role in the regulation of subcellular contractility by assembling into a ternary complex with Rho-kinase and Shroom2 in a phosphorylation-dependent manner. We propose that the KISS method is a comprehensive and useful substrate screen for various kinases.

Differential effects of putative protein kinase C inhibitors on contraction of rat aortic smooth muscle

1993 ◽  
Vol 264 (4) ◽  
pp. H1300-H1306 ◽  
Author(s):  
Y. Shimamoto ◽  
H. Shimamoto ◽  
C. Y. Kwan ◽  
E. E. Daniel
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Induced Response ◽  
Dependent Manner ◽  
Contractile Responses ◽  
Putative Protein ◽  
Calphostin C ◽  
Kinase C ◽  
Putative Protein Kinase

We investigated effects of three kinds of putative protein kinase C (PKC) inhibitors, calphostin C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and stauro-sporine, on aortic muscle contractions induced by KCl, phenylephrine, 12-O-tetradecanoylphorbol-13-acetate (TPA), and phorbol 12, 13-dibutyrate (PDBu). Calphostin C noncompetitively inhibited TPA-induced contractions in a concentration-dependent manner. At 10(-6) M, calphostin C completely abolished responses to TPA and also effectively inhibited PDBu-induced contractions. Such a concentration of calphostin C had no effect on KCl-induced contractions but decreased the maximal tension of phenylephrine-induced response curve by 35.3 +/- 6.6% H-7 (10(-5) M had little effect on TPA-induced contraction but significantly inhibited contractile responses to phenylephrine and KCl. Staurosporine (10(-8) M, 3 x 10(-8) M) inhibited contractile responses to KCl, phenylephrine, and TPA. We suggest that staurosporine and H-7, which are known to act on the catalytic domain of PKC carrying high degree of sequence homology with other protein kinases, are relatively nonselective for PKC. On the other hand, calphostin C acting on the regulatory domain of PKC, which is distinct from other protein kinases, may serve as a relatively more selective PKC inhibitor.

scholarly journals The MEK1 Scaffolding Protein MP1 Regulates Cell Spreading by Integrating PAK1 and Rho Signals

2005 ◽  
Vol 25 (12) ◽  
pp. 5119-5133 ◽  
Author(s):  
Ashok Pullikuth ◽  
Evangeline McKinnon ◽  
Hans-Joerg Schaeffer ◽  
Andrew D. Catling
Keyword(s):  
Rho Kinase ◽  
Biological Response ◽  
Cell Spreading ◽  
Scaffolding Protein ◽  
Dependent Manner ◽  
Scaffolding Proteins ◽  
Cellular Functions ◽  
Erk Activation ◽  
Extracellular Signal ◽  
Cytoskeletal Rearrangements

ABSTRACT How the extracellular signal-regulated kinase (ERK) cascade regulates diverse cellular functions, including cell proliferation, survival, and motility, in a context-dependent manner remains poorly understood. Compelling evidence indicates that scaffolding molecules function in yeast to channel specific signals through common components to appropriate targets. Although a number of putative ERK scaffolding proteins have been identified in mammalian systems, none has been linked to a specific biological response. Here we show that the putative scaffold protein MEK partner 1 (MP1) and its partner p14 regulate PAK1-dependent ERK activation during adhesion and cell spreading but are not required for ERK activation by platelet-derived growth factor. MP1 associates with active but not inactive PAK1 and controls PAK1 phosphorylation of MEK1. Our data further show that MP1, p14, and MEK1 serve to inhibit Rho/Rho kinase functions necessary for the turnover of adhesion structures and cell spreading and reveal a signal-channeling function for a MEK1/ERK scaffold in orchestrating cytoskeletal rearrangements important for cell motility.

scholarly journals Modulation of Calmodulin and Protein Kinase C Activities by Pencillium Mycotoxins

1999 ◽  
Vol 18 (2) ◽  
pp. 91-96 ◽  
Author(s):  
I. Pala ◽  
A. Srinivasan ◽  
P. J. S. Vig ◽  
D. Desaiah
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Complex Formation ◽  
Mammalian Brain ◽  
Dependent Manner ◽  
Active Conformation ◽  
Cellular Functions ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Ic50 Values

Calmodulin (CaM), a calcium-binding protein, is found in high concentrations in mammalian brain where it plays a pivotal role in a large number of cellular functions. Protein kinase C (PKC), a multifunctional cytosolic enzyme, in the presence of both Ca2+ and phospholipids, transduce extracellular signals into intracellu-lar events. Both CaM and PKC are partially involved in maintaining Ca2+ homeostasis in the cell. Any fluctuations in the intracel-lular Ca2+ can modulate cellular functions and may contribute to neuronal dysfunction. Hence, the present investigation was initiated to study the effects of some selected penicillium (naturally occurring tremorgenic) mycotoxins like secalonic acid, citreoviridin, and verruculogen on CaM activity, active conformation of CaM and PKC activity. Stimulation of CaM-deflcient bovine brain 3′-5′ phosphodieste rase (PDE) indicated CaM activity. The modification of CaM active conformation was studied by the binding of fluorescent probe N-phenyl-1-napthylamine (NPN) to CaM. Alterations in the fluorescence of dansyl-CaM was used to study the effect of these compounds on complex formation between CaM and PDE. Rat brain cytosolic PKC was studied using 32P-ATP as a measure of altered protein phosphorylation. The concentrations of mycotoxins used were in the range of 10 to 50 μM. All three mycotoxins inhibited CaM-stimulated PDE activity in a concentration-dependent manner. Citreoviridin and secalonic acid inhibited NPN fluorescence and Ca2+-dependent complex formation of dansyl-CaM and PDE. The IC50 values for NPN fluorescence of citreoviridin and secalonic acid were 13 μM and 19 μM respectively. However, verruculogen showed little effect on NPN fluorescence and the Ca2+-dependent complex formation of dansyl-CaM and PDE. These mycotoxins also inhibited PKC activity in a concentration-dependent manner with IC50 values of 19.8, 25.7, and 38.4 μM for secalonic acid, citreoviridin, and verruculogen, respectively. The results of our study suggest that these mycotoxins at very low concentrations are interacting with CaM and PKC. Such an effect could lead to impairment of neurotransmission and result in neurotoxicity.

Postal code for a plant MAPK

2012 ◽  
Vol 446 (2) ◽  
pp. e5-e7 ◽  
Author(s):  
Brian E. Ellis
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Mitogen Activated Protein Kinase ◽  
Postal Code ◽  
Biochemical Journal ◽  
Mitogen Activated Protein ◽  
Cellular Signal ◽  
Efficient Route ◽  
Kinase Substrates

Plants contain hundreds of protein kinases that are believed to provide cellular signal transduction services, but the identities of the proteins they are targeting are largely unknown. Using an Arabidopsis MAPK (mitogen-activated protein kinase) (MPK6) as a model, Sörensson et al. describe in this issue of the Biochemical Journal how arrayed combinatorial peptide scanning offers an efficient route to discovery of new potential kinase substrates.

Rho kinase mediates serum-induced contraction in fibroblast fibers independent of myosin LC20 phosphorylation

2003 ◽  
Vol 284 (3) ◽  
pp. C599-C606 ◽  
Author(s):  
Hiromi Nobe ◽  
Koji Nobe ◽  
Fabeha Fazal ◽  
Primal de Lanerolle ◽  
Richard J. Paul
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Kinase Inhibitor ◽  
Calf Serum ◽  
Rho Kinase ◽  
Fiber Formation ◽  
Dependent Manner ◽  
Sustained Contraction ◽  
Kinase C

Fibroblasts form fibers when grown in culture medium containing native type 1 collagen. The contractile forces generated can be precisely quantified and used to analyze the signal transduction pathways regulating fibroblast contraction. Calf serum (30%) induces a sustained contraction that is accompanied by a transient increase in intracellular calcium ([Ca2+]i). W-7, a calmodulin inhibitor, KN-62, an inhibitor of calcium/calmodulin-dependent protein kinase, and ML-7, a myosin light-chain kinase inhibitor, had no effects on either the contraction or the [Ca2+]i responses. Neither genistein, a tyrosine kinase inhibitor, nor calphostin C, a protein kinase C inhibitor, had major effects on force or [Ca2+]i. In contrast, the Rho kinase inhibitors (R)-(+)- trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) and HA1077 depressed the contraction in a dose-dependent manner without affecting the [Ca2+]iresponse. Stress fiber formation was also suppressed by Y-27632. Surprisingly, calf serum, Y-27632, and calf serum plus Y-27632 did not alter mono- or diphosphorylation of the myosin regulatory light chain (MRLC) compared with control untreated fibers. These results suggest that the sustained contraction of NIH 3T3 fibroblast fibers induced by calf serum is mediated by Rho kinase but is independent of a sustained increase in [Ca2+]i, calcium/calmodulin- or protein kinase C-dependent pathways, or increases in MRLC phosphorylation.

Involvement of Ca2+Sensitization in Ropivacaine-induced Contraction of Rat Aortic Smooth Muscle

2005 ◽  
Vol 103 (3) ◽  
pp. 548-555 ◽  
Author(s):  
Jingui Yu ◽  
Yasuyuki Tokinaga ◽  
Toshiyuki Kuriyama ◽  
Nobuhiko Uematsu ◽  
Kazuhiro Mizumoto ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Rho Kinase ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Membrane Translocation ◽  
Aortic Smooth Muscle ◽  
Calphostin C ◽  
Dose Dependent

Background The mechanisms of amino-amide local anesthetic agent-induced vasoconstriction remain unclear. The current study was designed to examine the roles of the protein kinase C (PKC), Rho kinase, and p44/42 mitogen-activated protein kinase (p44/42 MAPK) signaling pathways in calcium (Ca2+)-sensitization mechanisms in ropivacaine-induced vascular contraction. Methods Endothelium-denuded rat aortic rings, segments, and strips were prepared. The cumulative dose-response relations of contraction and intracellular Ca2+ concentration to ropivacaine were tested, using isometric force transducers and a fluorometer, respectively. The dose-dependent ropivacaine-induced phosphorylation of PKC and p44/42 MAPK and the membrane translocation of Rho kinase were also detected using Western blotting. Results Ropivacaine induced a dose-dependent biphasic contractile response and an increase in intracellular Ca2+ concentration of rat aortic rings, increasing at concentrations of 3 x 10 m to 3 x 10 m and decreasing from 10 m to 3 x 10 m, with a greater tension/intracellular Ca2+ concentration ratio than that induced with potassium chloride. The contraction was attenuated in a dose-dependent manner, by the PKC inhibitors bisindolylmaleimide I and calphostin C, the Rho-kinase inhibitor Y 27632, and the p44/42 MAPK inhibitor PD 098059. Ropivacaine also induced an increase in phosphorylation of PKC and p44/42 MAPK, and membrane translocation of Rho kinase in accordance with the contractile responses, which were also significantly inhibited by bisindolylmaleimide I and calphostin C, Y 27632, and PD 098059, correspondingly. Conclusion These findings demonstrated that PKC-, Rho kinase-, and p44/42 MAPK-mediated Ca2+-sensitization mechanisms are involved in the ropivacaine-induced biphasic contraction of rat aortic smooth muscle.

scholarly journals Functions and regulation of the serine/threonine protein kinase CK1 family: moving beyond promiscuity

2020 ◽  
Vol 477 (23) ◽  
pp. 4603-4621
Author(s):  
Luke J. Fulcher ◽  
Gopal P. Sapkota
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Therapeutic Targets ◽  
Signalling Pathways ◽  
Biological Processes ◽  
Cellular Functions ◽  
Intracellular Regulation ◽  
Constitutively Active

Regarded as constitutively active enzymes, known to participate in many, diverse biological processes, the intracellular regulation bestowed on the CK1 family of serine/threonine protein kinases is critically important, yet poorly understood. Here, we provide an overview of the known CK1-dependent cellular functions and review the emerging roles of CK1-regulating proteins in these processes. We go on to discuss the advances, limitations and pitfalls that CK1 researchers encounter when attempting to define relationships between CK1 isoforms and their substrates, and the challenges associated with ascertaining the correct physiological CK1 isoform for the substrate of interest. With increasing interest in CK1 isoforms as therapeutic targets, methods of selectively inhibiting CK1 isoform-specific processes is warranted, yet challenging to achieve given their participation in such a vast plethora of signalling pathways. Here, we discuss how one might shut down CK1-specific processes, without impacting other aspects of CK1 biology.

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