The C-kinase-activated protein phosphatase-1 (PP1) inhibitor of 17 kDa (CPI-17) is a specific inhibitor of the PP1 catalytic subunit (PP1c) and the myosin phosphatase (MP) holoenzyme. CPI-17 requires the phosphorylation of Thr38 in the peptide segment ³⁵ARV(P)TVKYDRREL⁴⁶ for inhibitory activity. CPI-17 regulates myosin phosphorylation in smooth muscle contraction and the tumorigenic transformation of several cell lines via the inhibition of MP. A phosphospecific antibody (anti-CPI-17^pThr38) against the phosphorylation peptide was used to determine the phosphorylation levels in cells. We found that phospho-CPI-17 and its closely related proteins are not present in HeLa and MCF7 cells after inducing phosphorylation by inhibiting phosphatases with calyculin A. In contrast, cross-reactions of proteins in the 40–220 kDa range with anti-CPI-17^pThr38 were apparent. Searching the protein database for similarities to the CPI-17 phosphorylation sequence revealed several proteins with 42–75% sequence identities. The LIMK2-1 isoform emerged as a possible PP1 inhibitor. Experiments with Flag-LIMK2-1 overexpressed in tsA201 cells proved that LIMK2-1 interacts with PP1c isoforms and is phosphorylated predominantly by protein kinase C. Phosphorylated LIMK2-1 inhibits PP1c and the MP holoenzyme with similar potencies (IC50 ~28–47 nM). In conclusion, our results suggest that LIMK2-1 is a novel phosphorylation-dependent inhibitor of PP1c and MP and may function as a CPI-17-like phosphatase inhibitor in cells where CPI-17 is present but not phosphorylated upon phosphatase inhibition.

Myosin phosphatase (MP) holoenzyme consists of protein phosphatase-1 (PP1) catalytic subunit (PP1c) associated with myosin phosphatase target subunit-1 (MYPT1) and it plays an important role in mediating the phosphorylation of the 20 kDa light chain (MLC20) of myosin, thereby regulating cell contractility. The association of MYPT1 with PP1c increases the phosphatase activity toward myosin; therefore, disrupting/dissociating this interaction may result in inhibition of the dephosphorylation of myosin. In this study, we probed how MYPT1^32–58 peptide including major PP1c interactive regions coupled with biotin and cell-penetrating TAT sequence (biotin-TAT-MYPT1) may influence MP activity. Biotin-TAT-MYPT1 inhibited the activity of MP holoenzyme and affinity chromatography as well as surface plasmon resonance (SPR) binding studies established its stable association with PP1c. Biotin-TAT-MYPT1 competed for binding to PP1c with immobilized GST-MYPT1 in SPR assays and it partially relieved PP1c inhibition by thiophosphorylated (on Thr696 and Thr853) MYPT1. Moreover, biotin-TAT-MYPT1 dissociated PP1c from immunoprecipitated PP1c-MYPT1 complex implying its holoenzyme disrupting ability. Biotin-TAT-MYPT1 penetrated into A7r5 smooth muscle cells localized in the cytoplasm and nucleus and exerted inhibition on MP with a parallel increase in MLC20 phosphorylation. Our results imply that the biotin-TAT-MYPT1 peptide may serve as a specific MP regulatory cell-penetrating peptide as well as possibly being applicable to further development for pharmacological interventions.