# LIMK2-1 Is a Phosphorylation-Dependent Inhibitor of Protein Phosphatase-1 Catalytic Subunit and Myosin Phosphatase Holoenzyme

## TL;DR

This study identifies LIMK2-1 as a new inhibitor of protein phosphatase-1 and myosin phosphatase, similar to CPI-17 but dependent on phosphorylation.

## Contribution

LIMK2-1 is newly identified as a phosphorylation-dependent inhibitor of PP1c and MP, functioning like CPI-17 in unphosphorylated CPI-17 contexts.

## Key findings

- LIMK2-1 interacts with PP1c and is phosphorylated by protein kinase C.
- Phosphorylated LIMK2-1 inhibits PP1c and MP with IC50 ~28–47 nM.
- LIMK2-1 may act as a CPI-17-like inhibitor in cells lacking phosphorylated CPI-17.

## Abstract

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-17pThr38) 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-17pThr38 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.