Regulation of Cellular Protein Phosphatase-1 (PP1) by Phosphorylation of the CPI-17 Family, C-kinase-activated PP1 Inhibitors

Abstract

. Here, the mechanisms underlying PP1 inhibition and the kinase/PP1 cross-talk mediated by CPI-17 and its related proteins, PHI, KEPI, and GBPI, are discussed.The reciprocal activities of protein kinases and phosphatases determine protein phosphorylation levels in cells. PP1 2 dephosphorylates phospho-Ser/Thr residues of proteins to regulate multiple signaling pathways at various cellular loci (1-3). Cellular PP1 is associated with PP1 regulatory proteins/subunits at their PP1-binding site, known as the RVXF motif. The binding of PP1 regulatory proteins thus confers substrate specificity and localization on cellular PP1. Nearly 100 polypeptides have been identified as PP1 regulatory proteins, and these account for the wide spectrum of PP1 function (1-3). In addition, eukaryotic cells express several PP1 inhibitor proteins that play important roles in regulating cellular PP1. The first generation of PP1 inhibitor proteins involves inhibitor-1, inhibitor-2, DARPP32, and NIPP-1, which potently inhibit the free catalytic subunit of PP1, but these inhibitor proteins were much less potent toward purified PP1 holoenzymes, MLCP and glycogen-bound PP1. Therefore, cellular PP1 holoenzymes were thought to undergo subunit dissociation prior to the inhibition of PP1 by the inhibitor proteins (1, 2). However, the number of PP1 holoenzymes that do undergo subunit dissociation in cells remains unclear.MLCP is a trimeric PP1 holoenzyme, consisting of a PP1∂ isoform and a regulatory complex of MYPT1 (aka MBS, M110) and a 21-kDa accessory subunit, and vital to control cellular phosphorylation in response to various signals (4). MYPT1 and PP1 bind through the MYPT1 KVKF segment, as well as its eight-repeat ankyrin motif at the N-terminal domain (5). Binding of the N-terminal 300-residue domain of MYPT1 is sufficient to allosterically regulate PP1 activity. The MYPT1 C-terminal domain directly binds to substrates, including myosin and ezrin/radixin/moesin (4). MLCP activity is reversibly regulated in response to various signals. For example, in smooth muscle, activation of the G-protein-coupled receptor inhibits MLCP, resulting in increased Ca 2ϩ sensitivity of myosin phosphorylation and contraction, whereas cyclic nucleotide signals can activate MLCP to induce smooth muscle relaxation (6). MLCP inhibition occurs upon MYPT1 phosphorylation at Thr 696 and Thr 853 (4). On the other hand, protein kinase G can activateMLCP(7).TheseregulatorysignalsareMYPT1isoform-dependent (8), suggesting an important role for MYPT1 in MLCP regulation. In addition, we identified the MLCP inhibitor protein, named CPI-17, which transduces G-protein signals into MLCP inhibition (9, 10). Based on sequence similarity, three CPI-17 homologs in the human genome, PHI, KEPI, and GBPI, were characterized as PP1 inhibitors (11-13). Each CPI-17 family member carries a PHIN domain, in which the sequences are Ͼ41% identical to CPI-17 (Fig. 1A). Indeed, all CPI-17 family members potently inhibit MLCP activity, which suggests new avenues for PP1 holoenzyme inhibition. ...

A Novel RhoA/ROCK-CPI-17-MEF2C Signaling Pathway Regulates Vascular Smooth Muscle Cell Gene Expression

“…In addition, the phosphatase activity of PP1␣ is regulated through interaction with specific inhibitor proteins like Inhibitor 1 and 2 (I1 and I2), and CPI-17. The potency of these inhibitor proteins is regulated by phosphorylation and dephosphorylation by cellular kinases and phosphatases, such as PKA, calcineurin, ROCK, and PKN (14,21,33). Our data demonstrates that phosphorylation of CPI-17 at Thr-38 by ROCK and/or PKN regulates PP1␣ ability to modulate gene expression; whereas, I1 and I2 have no effect on MEF2 transcriptional activity (13).…”

Inside-out Regulation of Ectodomain Cleavage of Cluster-of-Differentiation-44 (CD44) and of Neuregulin-1 Requires Substrate Dimerization

“…How can CD44S291 be dephosphorylated in response to TPA-dependent activation of PKC? PP1/2 serine phosphatase can indeed be activated by PKC and is regulated by endogenous PKC-activated inhibitors (8,41,46). molecules.…”

Integrin-linked kinase: Not so ‘pseudo’ after all

“…These include b1-integrin (Hannigan et al, 1996), myelin basic protein (Hannigan et al, 1996); b-parvin (Yamaji et al, 2001); glycogen synthase kinase-3b (GSK-3b) (Delcommenne et al, 1998); Akt (Persad et al, 2001); the regulatory light chain of myosin-II, MLC-20 ; the protein phosphatase-inhibitory proteins CPI-17, PHI-1 and KEPI (Erd + odi et al, 2003); the myosin-targeting subunit of the myosin (Attwell et al, 2003) Lactation, mammary differentiation (Akhtar et al, 2009) Smooth-muscle contraction; MLC-20 phosphorylation Eto, 2009) Mitotic spindle organization (Fielding et al, , 2011) Renal agenesis (Lange et al, 2009) Akt Ser473 phosphorylation, cell survival (Persad et al, 2000(Persad et al, , 2001) Zebrafish muscle fiber-ECM adhesion (Postel et al, 2008) Human cardiomyopathy (Knoll et al, 2007) (Zervas et al, 2001) Inhibition of growth, and apoptosis of anaplastic thyroid cancer cells (Younes et al, 2005) Pancreatic cancer xenograft apoptosis (Yau et al, 2005) Mammary tumor growth (White et al, 2001) Abbreviation: ILK, integrin-linked kinase. Evidence for kinase dependence is based on effects of point mutations predicted or shown to affect catalytic function, or inhibition by the QLT class of inhibitor, with or without corroborating genetic knockout/knockdown data.…”

“…Subsequently, by using solution and in-gel kinase assays, ILK was shown to directly phosphorylate MYPT1 (the myosin-targeting subunit of myosin lightchain phosphatase) (Mura´nyi et al, 2002) (Figure 1) and members of the CPI-17 family of protein phosphatase inhibitors Eto, 2009) (Figure 2), which had previously been shown to be phosphorylated at the same sites (Thr-38 of CPI-17, Thr-73 of KEPI and Thr-57 of PHI-1) by protein kinase-C (Eto, 2009). These phosphorylations convert the proteins from inactive to active phosphatase inhibitors.…”