Characterization of Ca2+/calmodulin-dependent protein kinase I as a myosin II regulatory light chain kinase in vitro and in vivo

Ca2+/calmodulin (CaM)-dependent protein kinase I (CaM-KI), which is a member of the multifunctional CaM-K family, is thought to be involved in various Ca2+-signalling pathways. In this report, we demonstrate that CaM-KI activated by an upstream kinase (CaM-K kinase), but not unactivated CaM-KI, phosphorylates myosin II regulatory light chain (MRLC) efficiently (Kcat, 1.7s-1) and stoichiometrically (0.8mol of phosphate/mol) in a Ca2+/CaM-dependent manner in vitro. One-dimensional phosphopeptide mapping and mutational analysis of MRLC revealed that the activated CaM-KI monophosphorylates only Ser-19 in MRLC. Transient expression of the Ca2+/CaM-independent form of CaM-KI (CaM-KI1-293) in HeLa cells induced Ser-19 phosphorylation of myosin, II accompanied by reorganization of actin filaments in the peripheral region of the cells. CaM-KI-induced reorganization of actin filaments was suppressed by co-expression of non-phosphorylatable MRLC mutants (S19A and T18AS19A). Furthermore, a kinase-negative form of CaM-KI (CaM-KI1-293,K49E) significantly reduced reorganization of actin filaments, indicating a dominant negative effect. This is the first demonstration that the activation of the CaM-KI cascade induces myosin II phosphorylation, resulting in regulation of actin filament organization in mammalian cells.

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Myosin Phosphatase and Cofilin Mediate cAMP/cAMP-dependent Protein Kinase-induced Decline in Endothelial Cell Isometric Tension and Myosin II Regulatory Light Chain Phosphorylation

Journal of Biological Chemistry ◽

10.1074/jbc.m503173200 ◽

2005 ◽

Vol 280 (38) ◽

pp. 33083-33095 ◽

Cited By ~ 37

Author(s):

Zoe M. Goeckeler ◽

Robert B. Wysolmerski

Keyword(s):

Endothelial Cell ◽

Protein Kinase ◽

Light Chain ◽

Myosin Ii ◽

Regulatory Light Chain ◽

Isometric Tension ◽

Myosin Phosphatase ◽

Dependent Protein Kinase ◽

Dependent Protein ◽

Regulatory Light Chain Phosphorylation

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Adrenocorticotrophic hormone stimulates phosphotyrosine phosphatase SHP2 in bovine adrenocortical cells: phosphorylation and activation by cAMP-dependent protein kinase

Biochemical Journal ◽

10.1042/bj3520483 ◽

2000 ◽

Vol 352 (2) ◽

pp. 483-490 ◽

Cited By ~ 25

Author(s):

Stéphane ROCCHI ◽

Isabelle GAILLARD ◽

Emmanuel VAN OBBERGHEN ◽

Edmond M. CHAMBAZ ◽

Isabelle VILGRAIN

Keyword(s):

Protein Kinase ◽

Kinase Assay ◽

Dependent Manner ◽

Adrenocorticotrophic Hormone ◽

Adrenocortical Cells ◽

Dependent Protein Kinase ◽

Phosphotyrosine Phosphatase ◽

Camp Dependent Protein Kinase ◽

Dependent Protein

During activation of adrenocortical cells by adrenocorticotrophic hormone (ACTH), tyrosine dephosphorylation of paxillin is stimulated and this correlates with protrusion of filopodial structures and a decreased number of focal adhesions. These effects are inhibited by Na3VO4, a phosphotyrosine phosphatase inhibitor [Vilgrain, Chinn, Gaillard, Chambaz and Feige (1998) Biochem. J. 332, 533–540]. However, the tyrosine phosphatases involved in these processes remain to be identified. In this study, we provide evidence that the Src homology domain (SH)2-containing phosphotyrosine phosphatase (SHP)2, but not SHP1, is expressed in adrenocortical cells and is phosphorylated upon ACTH challenge. ACTH (10-8M) treatment of 32P-labelled adrenocortical cells resulted in an increase in phosphorylated SHP2. By probing SHP2-containing immunoprecipitates with an antibody to phosphoserine we found that SHP2 was phosphorylated on serine in ACTH-treated cells in a dose- and time-dependent manner. Furthermore, using an in vitro kinase assay, we showed that SHP2 was a target for cAMP-dependent protein kinase (PKA). Serine was identified as the only target amino acid phosphorylated in SHP2. Phosphorylation of SHP2 by PKA resulted in a dramatic stimulation of phosphatase activity measured either with insulin receptor substrate-1 or with the synthetic peptide [32P]poly(Glu/Tyr) as substrate. In an in-gel assay of SHP2-containing immunoprecipitates, phosphorylated in vitro by PKA or isolated from adrenocortical cells treated with 10nM ACTH, a pronounced activation of SHP2 activity was shown. These observations clearly support the idea that a PKA-mediated signal transduction pathway contributes to SHP2 regulation in adrenocortical cells and point to SHP2 as a possible mediator of the effects of ACTH.

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ATR-Dependent Phosphorylation of DNA-Dependent Protein Kinase Catalytic Subunit in Response to UV-Induced Replication Stress

Molecular and Cellular Biology ◽

10.1128/mcb.00048-06 ◽

2006 ◽

Vol 26 (20) ◽

pp. 7520-7528 ◽

Cited By ~ 92

Author(s):

Hirohiko Yajima ◽

Kyung-Jong Lee ◽

Benjamin P. C. Chen

Keyword(s):

Protein Kinase ◽

Uv Irradiation ◽

Cellular Response ◽

Replication Stress ◽

Dominant Negative ◽

Catalytic Subunit ◽

Dependent Manner ◽

Dependent Protein Kinase ◽

Dependent Protein

ABSTRACT Phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) upon ionizing radiation (IR) is essential for cellular radioresistance and nonhomologous-end-joining-mediated DNA double-strand break repair. In addition to IR induction, we have previously shown that DNA-PKcs phosphorylation is increased upon camptothecin treatment, which induces replication stress and replication-associated double-strand breaks. To clarify the involvement of DNA-PKcs in this process, we analyzed DNA-PKcs phosphorylation in response to UV irradiation, which causes replication stress and activates ATR (ATM-Rad3-related)/ATM (ataxia-telangiectasia mutated) kinases in a replication-dependent manner. Upon UV irradiation, we observed a rapid DNA-PKcs phosphorylation at T2609 and T2647, but not at S2056, distinct from that induced by IR. UV-induced DNA-PKcs phosphorylation occurs specifically only in replicating cells and is dependent on ATR kinase. Inhibition of ATR activity via caffeine, a dominant-negative kinase-dead mutant, or RNA interference led to the attenuation of UV-induced DNA-PKcs phosphorylation. Furthermore, DNA-PKcs associates with ATR in vivo and is phosphorylated by ATR in vitro, suggesting that DNA-PKcs could be the direct downstream target of ATR. Taken together, these results strongly suggest that DNA-PKcs is required for the cellular response to replication stress and might play an important role in the repair of stalled replication forks.

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