Background: The Myosin Phosphatase (MP) holoenzyme is composed of a Protein Phosphatase type 1 (PP1) catalytic subunit and a regulatory subunit termed Myosin Phosphatase Target subunit 1 (MYPT1). Besides dephosphorylation of myosin, MP has been implicated in the control of cell proliferation via dephosphorylation and activation of the tumor suppressor gene products, retinoblastoma protein (pRb) and merlin. Inhibition of MP was shown to attenuate the drug-induced cell death of leukemic cells by chemotherapeutic agents, while activation of MP might have a sensitizing effect. Objective: Recently, Epigallocatechin-Gallate (EGCG), a major component of green tea, was shown to activate MP by inducing the dephosphorylation of MYPT1 at phospho-Thr696 (MYPT1pT696), which might confer enhanced chemosensitivity to cancer cells. Methods: THP-1 leukemic cells were treated with EGCG and Daunorubicin (DNR) and cell viability was analyzed. Phosphorylation of tumor suppressor proteins was detected by Western blotting. Results: EGCG or DNR (at sub-lethal doses) alone had moderate effects on cell viability, while the combined treatment caused a significant decrease in the number of viable cells by enhancing apoptosis and decreasing proliferation. EGCG plus DNR decreased the phosphorylation level of MYPT1pT696, which was accompanied by prominent dephosphorylation of pRb. In addition, significant dephosphorylation of merlin was observed when EGCG and DNR were applied together. Conclusion: Our results suggest that EGCG-induced activation of MP might have a regulatory function in mediating the chemosensitivity of leukemic cells via dephosphorylation of tumor suppressor proteins.

Monocyte to macrophage differentiation is characterized by the activation of various signal transduction pathways, which may be modulated by protein phosphorylation; however, the impact of protein kinases and phosphatases is not well understood yet. It has been demonstrated that actomyosin rearrangement during macrophage differentiation is dependent on Rho-associated protein kinase (ROCK). Myosin phosphatase (MP) target subunit-1 (MYPT1) is one of the major cellular substrates of ROCK, and MP is often a counter enzyme of ROCK; therefore, MP may also control macrophage differentiation. Changes in MP activity and the effects of MP activation were studied on PMA or l,25(OH)₂D₃-induced differentiation of monocytic THP-1 cells. During macrophage differentiation, phosphorylation of MYPT1 at Thr696 and Thr853 increased significantly, resulting in inhibition of MP. The ROCK inhibitor H1152 and the MP activator epigallocatechin-3-gallate (EGCG) attenuated MYPT1 phosphorylation and concomitantly decreased the extent of phosphorylation of 20 kDa myosin light chain. H1152 and EGCG pretreatment also suppressed the expression of CD11b and weakened the PMA-induced adherence of the cells. Our results indicate that MP activation/inhibition contributes to the efficacy of monocyte to macrophage differentiation, and this enzyme may be a target for pharmacological interventions in the control of disease states that are affected by excessive macrophage differentiation.

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.