Uncoupling of GPCR and RhoA‐induced Ca²⁺‐sensitization of chicken amnion smooth muscle lacking CPI‐17

Abstract

Ca2+ -sensitization of smooth muscle occurs through inhibition of myosin light chain phosphatase (MLCP) leading to an increase in the MLCK:MLCP activity ratio. MLCP is inhibited through phosphorylation of its regulatory subunit (MYPT-1) following activation of the RhoA/Rho kinase (ROK) pathway or through phosphorylation of the PP1c inhibitory protein, CPI-17, by PKC d or ROK. Here, we explore the crosstalk between these two modes of MLCP inhibition in a smooth muscle of a natural CPI-17 knockout, chicken amnion. GTPcS elicited Ca 2+ -sensitized force which was relaxed by GDI or Y-27632, however, U46619, carbachol and phorbol ester failed to induce Ca 2+ -sensitized force, but were rescued by recombinant CPI-17, and were sensitive to Y-27632 inhibition. In the presence, but not absence, of CPI-17, U46619 also significantly increased GTP Á RhoA. There was no affect on MYPT-1 phosphorylation at T695, however, T850 phosphorylation increased in response to GTPcS stimulation. Together, these data suggest a role for CPI-17 upstream of RhoA activation possibly through activation of another PP1 family member targeted by CPI-17.

Rat pulmonary arterial smooth muscle myosin light chain kinase and phosphatase activities decrease with age

“…This speculation is in keeping with the observation that Rho-kinase is important for lung morphogenesis during fetal life (24). Aside from the RhoA/Rho-kinase, inhibition of the MLCP has been reported to occur through CPI-17-dependent phosphorylation via PKC␦ (31). This inhibitory pathway is independent of the Rho/Rhokinase but is believed to only play a minor role in the G protein-coupled mechanism of Ca 2ϩ sensitization (30).…”

RhoA-Rho kinase pathway mediates thrombin- and U-46619-induced phosphorylation of a myosin phosphatase inhibitor, CPI-17, in vascular smooth muscle cells

“…In line with this possibility, recent reports have shown that the contractility of cultured VSMCs display a greater dependency than fresh tissue on the RhoA-ROCK pathway (3). Moreover, the phosphorylation site on MYPT1 changes upon culturing (53). Mature VSMCs are in a quiescent contractile phenotype under physiological conditions.…”

Maternal Phosphatase Inhibitor-2 Is Required for Proper Chromosome Segregation and Mitotic Synchrony During Drosophila Embryogenesis

“…For example, in vertebrates, myosin phosphatase (MYPT1-PP1) is selectively inhibited by the protein CPI-17, which does not inhibit other holoenzyme forms of PP1, such as glycogen-associated phosphatase (GM-PP1) . Phosphorylation of Thr38 in CPI-17 increases its inhibitory potency .1000-fold (Eto et al 1999), and smooth muscle contracts in response to hormones that trigger CPI-17 phosphorylation in a process called calcium sensitization (Stevenson et al 2004). Inhibitor-2 (I-2) is the most ancient of the PP1 inhibitor proteins and is conserved among all eukaryotes, from GLC8 in yeast to I-2 in Caenorhabditis elegans, Drosophila, Xenopus, and humans (Gruppuso et al 1985;Roach et al 1985;Tunget al 1995;Li et al 2007).…”