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Phosphorylation-induced conformational switching of CPI-17 produces a potent myosin phosphatase inhibitor.

ABSTRACT: Phosphorylation of endogenous inhibitor proteins for type-1 Ser/Thr phosphatase (PP1) provides a mechanism for reciprocal coordination of kinase and phosphatase activities. A myosin phosphatase inhibitor protein CPI-17 is phosphorylated at Thr38 through G-protein-mediated signals, resulting in a >1000-fold increase in inhibitory potency. We show here the solution NMR structure of phospho-T38-CPI-17 with rmsd of 0.36 +/- 0.06 A for the backbone secondary structure, which reveals how phosphorylation triggers a conformational change and exposes an inhibitory surface. This active conformation is stabilized by the formation of a hydrophobic core of intercalated side chains, which is not formed in a phospho-mimetic D38 form of CPI-17. Thus, the profound increase in potency of CPI-17 arises from phosphorylation, conformational change, and hydrophobic stabilization of a rigid structure that poses the phosphorylated residue on the protein surface and restricts its hydrolysis by myosin phosphatase. Our results provide structural insights into transduction of kinase signals by PP1 inhibitor proteins.

SUBMITTER: Eto M

PROVIDER: S-EPMC2217667 | biostudies-literature | 2007 Dec

REPOSITORIES: biostudies-literature

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Phosphorylation-induced conformational switching of CPI-17 produces a potent myosin phosphatase inhibitor.

Eto Masumi M Kitazawa Toshio T Matsuzawa Fumiko F Aikawa Sei-Ichi S Kirkbride Jason A JA Isozumi Noriyoshi N Nishimura Yumi Y Brautigan David L DL Ohki Shin-Ya SY

Structure (London, England : 1993) 20071201 12

Phosphorylation of endogenous inhibitor proteins for type-1 Ser/Thr phosphatase (PP1) provides a mechanism for reciprocal coordination of kinase and phosphatase activities. A myosin phosphatase inhibitor protein CPI-17 is phosphorylated at Thr38 through G-protein-mediated signals, resulting in a >1000-fold increase in inhibitory potency. We show here the solution NMR structure of phospho-T38-CPI-17 with rmsd of 0.36 +/- 0.06 A for the backbone secondary structure, which reveals how phosphorylati ...[more]

PMID: 18073109

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Project description:The Cpi-17 (ppp1r14) gene family is an evolutionarily conserved, vertebrate specific group of protein phosphatase 1 (PP1) inhibitors. When phosphorylated, Cpi-17 is a potent inhibitor of myosin phosphatase (MP), a holoenzyme complex of the regulatory subunit Mypt1 and the catalytic subunit PP1. Myosin phosphatase dephosphorylates the regulatory myosin light chain (Mlc2) and promotes actomyosin relaxation, which in turn, regulates numerous cellular processes including smooth muscle contraction, cytokinesis, cell motility, and tumor cell invasion. We analyzed zebrafish homologs of the Cpi-17 family, to better understand the mechanisms of myosin phosphatase regulation. We found single homologs of both Kepi (ppp1r14c) and Gbpi (ppp1r14d) in silico, but we detected no expression of these genes during early embryonic development. Cpi-17 (ppp1r14a) and Phi-1 (ppp1r14b) each had two duplicate paralogs, (ppp1r14aa and ppp1r14ab) and (ppp1r14ba and ppp1r14bb), which were each expressed during early development. The spatial expression pattern of these genes has diverged, with ppp1r14aa and ppp1r14bb expressed primarily in smooth muscle and skeletal muscle, respectively, while ppp1r14ab and ppp1r14ba are primarily expressed in neural tissue. We observed that, in in vitro and heterologous cellular systems, the Cpi-17 paralogs both acted as potent myosin phosphatase inhibitors, and were indistinguishable from one another. In contrast, the two Phi-1 paralogs displayed weak myosin phosphatase inhibitory activity in vitro, and did not alter myosin phosphorylation in cells. Through deletion and chimeric analysis, we identified that the difference in specificity for myosin phosphatase between Cpi-17 and Phi-1 was encoded by the highly conserved PHIN (phosphatase holoenzyme inhibitory) domain, and not the more divergent N- and C- termini. We also showed that either Cpi-17 paralog can rescue the knockdown phenotype, but neither Phi-1 paralog could do so. Thus, we provide new evidence about the biochemical and developmental distinctions of the zebrafish Cpi-17 protein family.

Dataset Information

Phosphorylation-induced conformational switching of CPI-17 produces a potent myosin phosphatase inhibitor.

Publications

Phosphorylation-induced conformational switching of CPI-17 produces a potent myosin phosphatase inhibitor.

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