Phosphorylation of CPI17 and myosin binding subunit of type 1 protein phosphatase by p21-activated kinase

doi:10.1016/S0006-291X(02)02302-1

Biochemical and Biophysical Research Communications

Volume 297, Issue 4, 4 October 2002, Pages 773-778

https://doi.org/10.1016/S0006-291X(02)02302-1 Get rights and content

Abstract

CPI17 and myosin binding subunit of type 1 protein phosphatase (MBS) are the regulators of myosin light chain phosphatase (MLCP). The function of both regulators is controlled by phosphorylation. The phosphorylation of CPI17 at Thr38 significantly enhances the inhibitory activity of CPI17 and the phosphorylation at Thr641 of MBS decreases the MLCP activity. Here, we found that p21-activated protein kinase (PAK) phosphorylates both CPI17 at Thr38 and MBS at Thr641. For CPI17, PAK specifically phosphorylated at Thr38, since the mutation of Thr38 to Ala completely abolished the phosphorylation. On the other hand, PAK phosphorylated Thr641 but not Thr799 of MBS, the site phosphorylated by Rho kinase. Because PAK phosphorylates MBS more than 1 mol/mol, it is anticipated that PAK also phosphorylates other sites in addition to Thr641. CPI17 phosphorylation induced by PAK significantly enhanced the inhibitory activity of CPI17. On the other hand, the phosphorylation of MBS by PAK also decreased the MLCP activity. These results raise the possibility that the PAK pathway plays a role in MLCP regulation.

Section snippets

Materials and methods

Chemicals. [γ-³²P]ATP (3000 Ci/mmol) was obtained from Du Pont-NEN; BSA, Microcystin LR and ATP-γ-S were from Sigma Chemical. All other chemicals were of reagent grade.

Production of MLCP and PP1δconstructs. A SalI site was created at the 5^′-side of the initiation codon of rat MBS cDNA. The cDNA encoding entire coding region of rat MBS was excised and subcloned into pFastbac HTb baculovirus transfer vector (Invitrogen, CA). The recombinant virus was then produced according to manufacturer’s

Results and discussion

Fig. 1A shows the time course of phosphorylation of CPI17 by PAK. CPI17 was rapidly phosphorylated in the conditions used to 1.0±0.1 mol/mol. It is known that the phosphorylation of CPI17 at Thr38 significantly enhances the inhibitory activity of CPI17 [19], [21], [22]. Therefore, we examined it to see whether PAK phosphorylates CPI17 at Thr38 by using the antibodies specifically recognizing the phosphorylated Thr38 as probes. Anti-P-Thr38 antibodies recognized the CPI17 phosphorylated by PAK

Acknowledgements

We thank Dr. P. Cohen (University of Dundee), Dr. T. Leung (University of Singapore), and Dr. M. Yazawa (Hokkaiddo University) for generous gifts of rat1 MBS cDNA, GST-PAK1, and porcine CPI17 cDNA, respectively. We also thank Dr. K. Kikuchi (Hokkaiddo University) for rat PP1δ cDNA and rabbit anti-rat PP1δ antibodies. This work was supported by NIH Grants HL60831 and HL61426.

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^☆: Abbreviations: MLC, myosin light chain; MLCK, myosin light chain kinase; PP1, type 1 protein phosphatase; MLCP, myosin light chain phosphatase; MBS, myosin binding subunit of PP1; PP1c, catalytic subunit of PP1; PAK, p21-activated protein kinase; PKC, protein kinase C; 2-ME, 2-mercaptoethanol.

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Phosphorylation of CPI17 and myosin binding subunit of type 1 protein phosphatase by p21-activated kinase☆

Abstract

Section snippets

Materials and methods

Results and discussion

Acknowledgements

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