Phosphorylation of endogenous inhibitor proteins for type-1 Ser/Thr phosphatase (PP1) provides a mechanism for reciprocal coordination of kinase and phosphatase activities. A myosin phosphatase inhibitor protein CPI-17 is phosphorylated at Thr38 through G-protein-mediated signals, resulting in a >1000-fold increase in inhibitory potency. We show here the solution NMR structure of phospho-T38-CPI-17 with rmsd of 0.36 +/- 0.06 A for the backbone secondary structure, which reveals how phosphorylation triggers a conformational change and exposes an inhibitory surface. This active conformation is stabilized by the formation of a hydrophobic core of intercalated side chains, which is not formed in a phospho-mimetic D38 form of CPI-17. Thus, the profound increase in potency of CPI-17 arises from phosphorylation, conformational change, and hydrophobic stabilization of a rigid structure that poses the phosphorylated residue on the protein surface and restricts its hydrolysis by myosin phosphatase. Our results provide structural insights into transduction of kinase signals by PP1 inhibitor proteins.

Dephosphorylation of actin-binding proteins by a specialized form of protein Ser/Thr phosphatase type-1 (PP1) regulates smooth muscle contraction and morphology and motility of nonmuscle cells. This myosin and ezrin/radixin/moesin (ERM)-targeted phosphatase comprises the delta isoform PP1 catalytic subunit plus a primary regulatory subunit called myosin phosphatase targeting (MYPT1). We reconstructed myosin/ERM phosphatase in living rat embryo fibroblasts (REF52 cells) by transient expression of epitope-tagged MYPT1 (myc-MYPT1) plus HA-tagged PP1. Unexpectedly, wild-type myc-MYPT1 expressed alone accumulated predominantly in the nucleus, as visualized by immunofluorescent microscopy, whereas if coexpressed with HA-PP1, it was localized in the cytosol and deposited on cytoskeleton myofilaments. The F38A mutation of MYPT1 that eliminates PP1 binding gave nuclear localization of myc-MYPT1, even when coexpressed with HA-PP1. Thus, expression of both subunits was necessary to form myosin/ERM phosphatase in situ and mediate myofilament localization. The results indicate there is little endogenous PP1 available for interaction or interchange with ectopic regulatory subunits in living cells. We concluded that myosin binding by the C-terminal domain of MYPT1 is not sufficient to override nuclear import in fibroblasts, but the binding of PP1 to myc-MYPT1 neutralizes nuclear import. Full-length myc-MYPT1 plus HA-PP1 induced only subtle changes in organization of the actin cytoskeleton, however coexpression of myc-MYPT1(1-300) with HA-PP1 dispersed stress fibers without major alteration in morphology and myc-MYPT1(1-498) disrupted the cytoskeleton and produced radically extended cells that appeared like neurons. Based on these responses, we conclude that the MYPT1 C-terminus functions as an auto-inhibitory domain, and a central domain in MYPT1 can mediate extensive reorganization of the actin cytoskeleton.

CPI-17 (C-kinase-activated protein phosphatase-1 (PP1) inhibitor, 17kDa) is a cytoplasmic protein predominantly expressed in mature smooth muscle (SM) that regulates the myosin-associated PP1 holoenzyme (MLCP). Here, we show CPI-17 expression in proliferating cells, such as pancreatic cancer and hyperplastic SM cells. Immunofluorescence showed that CPI-17 was concentrated in nuclei of human pancreatic cancer (Panc1) cells. Nuclear accumulation of CPI-17 was also detected in the proliferating vascular SM cell culture and cells at neointima of rat vascular injury model. The N-terminal 21-residue tail domain of CPI-17 was necessary for the nuclear localization. Phospho-mimetic Asp-substitution of CPI-17 at Ser12 attenuated the nuclear import. CPI-17 phosphorylated at Ser12 was not localized at nuclei, suggesting a suppressive role of Ser12 phosphorylation in the nuclear import. Activated CPI-17 bound to all three isoforms of PP1 catalytic subunit in Panc1 nuclear extracts. CPI-17 knockdown in Panc1 resulted in dephosphorylation of histone H3 at Thr3, Ser10 and Thr11, whereas it had no effects on the phosphorylation of myosin light chain and merlin, the known targets of MLCP. In parallel, CPI-17 knockdown suppressed Panc1 proliferation. We propose that CPI-17 accumulated in the nucleus through the N-terminal tail targets multiple PP1 signaling pathways regulating cell proliferation.

Focal adhesions attach cultured cells to the extracellular matrix, and we found endogenous protein phosphatase-1␣ isoform (PP1␣) localized in adhesions across the entire area of adherent fibroblasts. However, in fibroblasts migrating into a scrape wound or spreading after replating PP1␣ did not appear in adhesions near the leading edge but was recruited into other adhesions coincident in time and space with incorporation of tensin. Endogenous tensin and PP1␣ co-precipitated from cell lysates with isoform-specific PP1 antibodies. Chemical crosslinking of focal adhesion preparations with Lomant's reagent demonstrated molecular proximity of endogenous PP1␣ and tensin, whereas neither focal adhesion kinase nor vinculin was cross-linked and co-precipitated with PP1␣, suggesting distinct spatial subdomains within adhesions. Transient expression of truncated tensin showed the N-terminal 360 residues, which comprise a protein-tyrosine phosphatase domain, alone were sufficient for isoform-selective co-precipitation of co-expressed PP1␣. Human prostate cancer PC3 cells are deficient in tensin relative to fibroblasts and have fewer, mostly peripheral adhesions. Transient expression of green fluorescent protein tensin in these cancer cells induced formation of adhesions and recruited endogenous PP1␣ into those adhesions. Thus, the protein-tyrosine phosphatase domain of tensin exhibits isoform-specific association with PP1␣ in a restricted spatial region of adhesions that are formed during cell migration.Focal adhesions link force-generating elements of the actin cytoskeleton to the extracellular matrix. These adhesions are assembled initially in response to integrin engagement as cells protrude their leading edge in the process of spreading and migration (1-3). These initial adhesions contain focal adhesion kinase, paxillin, vinculin, and other proteins (4, 5). Phosphorylation of these proteins on Tyr residues accompanies the assembly of these adhesions and P-Tyr 2 immunostaining is concentrated at these sites (6). Focal adhesions dynamically exchange protein components as they connect to F-actin filaments and develop contractile force for cell locomotion. Adhesions that produce actomyosin-dependent movement of fibronectin receptors have been called "fibrillar adhesions" (7) to distinguish them from adhesions near the leading edge of the cell. These adhesions are relatively enriched in tensin, a protein that has a protein-tyrosine phosphatase domain related to PTEN (phosphatase and tensin1 homologue on chromosome 10), an Src homology 2 (SH2) domain, and a phosphotyrosine binding (PTB) domain (8, 9). The overall process of focal adhesion maturation can be followed either in cells migrating on two-dimensional surfaces or after re-plating onto matrix-coated surfaces as cells spread. It is now appreciated that focal adhesions are dynamic structures rapidly exchanging constituent proteins (10). Current work on the dynamics of focal adhesions suggests that Tyr phosphorylation is a major requirement for their turnover. However, re...