Dual Ser and Thr phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by MYPT-associated kinase

Phosphorylation of CPI-17 and PHI-1 by the MYPT1-associated kinase (M110 kinase) was investigated. M110 kinase is a recently identified serine/threonine kinase with a catalytic domain that is homologous to that of ZIP kinase (ZIPK. GST-rN-ZIPK, a constitutively active GST fusion fragment, phosphorylates CPI-17 (but not PHI-1) to a stoichiometry of 1.7 mol/mol. Phosphoamino acid analysis revealed phosphorylation of both Ser and Thr residues. Phosphorylation sites in CPI-17 were identified as Thr 38 and Ser 12 using Edman sequencing with (32)P release and a point mutant of Thr 38.     

Phosphorylation of CPI-17, an inhibitory phosphoprotein of smooth muscle myosin phosphatase, by Rho-kinase

Phosphorylation of CPI-17 by Rho-associated kinase (Rho-kinase) and its effect on myosin phosphatase (MP) activity were investigated. CPI-17 was phosphorylated by Rho-kinase to 0.92 mol of P/mol of CPI-17 in vitro. The inhibitory phosphorylation site was Thr(38) (as reported previously) and was identified using a point mutant of CPI-17 and a phosphorylation state-specific antibody. Phosphorylation by Rho-kinase dramatically increased the inhibitory effect of CPI-17 on MP activity. Thus, CPI-17 as a substrate of Rho-kinase could be involved in the Ca(2+) sensitization of smooth muscle contraction as a downstream effector of Rho-kinase.     

Identification of human CPI-17, an inhibitory phosphoprotein for myosin phosphatase

CPI-17 is a phosphorylation-dependent inhibitor of myosin phosphatase. cDNA clones encoding CPI-17 were isolated from a human aorta library. Overlapping clones indicated two isoforms: CPI-17alpha was 147 residues and mass of 16.7 kDa; CPI-17beta (120 residues, mass 13.5 kDa) resulted from a deletion in the alpha-isoform of 27 residues, sequence 68-94. N-terminal 67 residues of all CPI-17 isoforms (human, porcine, rat and mouse) were highly conserved (for the human and porcine isoforms the identity was 91%). The presence of the two human isoforms was detected from cDNA sequences amplified by RT-PCR and by Western blots on human aorta. The cloned human CPI-17 gene indicated 4 coding exons and CPI-17beta was an alternative splice variant due to deletion of the second exon. FISH analysis located the human CPI-17 gene on chromosome 19q13.1.     

Cerebellar long-term synaptic depression requires PKC-mediated activation of CPI-17, a myosin/moesin phosphatase inhibitor

Cerebellar LTD requires brief activation of PKC and is expressed as a functional downregulation of AMPA receptors. Modulation of vascular smooth-muscle contraction by G protein-coupled receptors (called Ca(2+) sensitization) also involves PKC phosphorylation and activation of a specific inhibitor of myosin/moesin phosphatase (MMP). This inhibitor, called CPI-17, is also expressed in brain. Here, we tested the hypothesis that LTD, like Ca(2+) sensitization, employs a PKC/CPI-17 cascade. Introduction of activated recombinant CPI-17 into cells produced a use-dependent attenuation of glutamate-evoked responses and occluded subsequent LTD. Moreover, the requirement for endogenous CPI-17 in LTD was demonstrated with neutralizing antibodies plus gene silencing by siRNA. These interventions had no effect on basal synaptic strength but blocked LTD induction. Thus, a biochemical circuit that involves PKC-mediated activation of CPI-17 modulates the distinct physiological processes of vascular contractility and cerebellar LTD.     

Novel in vitro and in vivo phosphorylation sites on protein phosphatase 1 inhibitor CPI-17

CPI-17 is a protein phosphatase 1 (PP1) inhibitor that has been shown to act on the myosin light chain phosphatase. CPI-17 is phosphorylated on Thr-38 in vivo, thus enhancing its ability to inhibit PP1. Thr-38 has been shown to be the target of several protein kinases in vitro. Originally, the expression of CPI-17 was proposed to be smooth muscle specific. However, it has recently been found in platelets and we show in this report that it is endogenously phosphorylated in brain on Ser-128 in a domain unique to CPI-17. Ser-128 is within a consensus phosphorylation site for protein kinase A (PKA) and calcium calmodulin kinase II. However, these two kinases do not phosphorylate Ser-128 in vitro but phosphorylate Ser-130 and Thr-38, respectively. The kinase responsible for Ser-128 phosphorylation remains to be identified. CPI-17 has strong sequence similarity with PHI-1 (which is also a phosphatase inhibitor) and LimK-2 kinase. The novel in vivo and in vitro phosphorylation sites (serines 128 and 130) are in a region/domain unique to CPI-17, suggesting a specific interaction domain that is regulated by phosphorylation.     

Phosphorylation of bovine platelet myosin by protein kinase C

Bovine platelet myosin is phosphorylated by protein kinase C at multiple sites. Most of the phosphate is incorporated in the 20,000-dalton light chain although some phosphate is incorporated in the heavy chain. Phosphorylation of the 20,000-dalton light chain of platelet myosin is 10 times faster than the phosphorylation of smooth muscle myosin. Platelet myosin light chain is first phosphorylated at a threonine residue followed by a serine residue. Dominant phosphorylation sites of the 20,000-dalton light chain are estimated as serine-1, serine-2, and threonine-9. Prolonged phosphorylation by protein kinase C resulted in an additional phosphorylation site which, on the basis of limited proteolysis, appears to be either serine-19 or threonine-18. Phosphorylation by protein kinase C causes an inhibition of actin-activated ATPase activity of platelet myosin prephosphorylated by myosin light chain kinase. Inhibition of ATPase activity is due to a decreased affinity of myosin for actin, and no change in Vmax is observed. It is shown that platelet myosin also exhibits the 6S to 10S conformation transition as judged by viscosity and gel filtration methods. Mg2(+)-ATPase activity of platelet myosin is paralleled with the 10S-6S transition. Phosphorylation by protein kinase C affects neither the 10S-6S transition nor the myosin filament formation. Therefore, the inhibition of actin-activated ATPase activity of platelet myosin is not due to the change in the myosin conformation.     

Phosphorylation of myosin phosphatase targeting subunit 3 (MYPT3) and regulation of protein phosphatase 1 by protein kinase A

Myosin phosphatase targeting subunit 3 (MYPT3) and transforming growth factor-beta-inhibited membrane-associated protein (TIMAP) are two closely related myosin-binding targeting subunits of protein phosphatase 1 (PP1c) with a characteristic CAAX (where AA indicates aliphatic amino acid) box at the C termini. Here we show that MYPT3 can be a substrate for protein kinase A (PKA). We first mapped the multiple phosphorylation sites within a central conserved motif. Deletion or mutations of this motif resulted in enhancement of the associated PP1c activity, suggesting that phosphorylation of MYPT3 may play an important role in regulating PP1c catalytic activity. However, unlike the other known MYPTs, which upon phosphorylation inhibit PP1c, PKA phosphorylation of MYPT3 resulted in PP1c activation, indicating a different mode of action. There is a direct interaction between the central conserved phosphorylated site motif with the N-terminal ankyrin repeat region; this interaction was significantly reduced with MYPT3 phosphorylation or acidic phosphorylation site mutations, with concomitant alterations in biochemical and morphological consequences. We therefore propose a novel mechanism for the phosphorylation of MYPT3 by PKA and activation of the catalytic activity through direct interaction of a central region of MYPT3 with its N-terminal region.     

Inhibition of myosin/moesin phosphatase by expression of the phosphoinhibitor protein CPI-17 alters microfilament organization and retards cell spreading

Cell migration and cytokinesis require reorganization of the cytoskeleton, involving phosphorylation and dephosphorylation of proteins such as myosin II and moesin. Myosin and moesin bind directly to a regulatory subunit of myosin/moesin phosphatase (MMP) that contains a protein type-1 phosphatase (PP1) catalytic subunit. Here we examined the role of MMP in cytoskeletal dynamics using a phosphorylation-dependent inhibitor protein specific for MMP, called CPI-17. Fibroblasts do not express CPI-17, making them a null background to study effects of expression. Wild type CPI-17 in rat embryo fibroblasts caused (1) abnormal accumulation of cortical F-actin fibers, distinct from the stress fibers induced by expression of active RhoA; (2) progressive contraction of cell area, leaving behind filamentous extensions that stained for F-actin and moesin, but not myosin; and (3) significantly retarded spreading of fibroblasts on fibronectin with elevated myosin II light chain phosphorylation. A phosphorylation site mutant CPI-17(T38A) and inhibitor-2 (Inh2), another PP1-specific inhibitor protein, served as controls and did not elicit these same responses when expressed at the same level as CPI-17. Inhibition of myosin light chain kinase by ML-9 prevented the abnormal accumulation of cortical microfilaments by CPI-17, but did not reverse shrinkage in area, whereas kinase inhibitors HA1077 and H7 prevented CPI-17-induced changes in microfilament distribution and cell contraction. These results highlight the physiological importance of myosin/moesin phosphatase regulation to dynamic remodeling of the cytoskeleton.     

Association of CPI-17 with protein kinase C and casein kinase I

The protein kinase C-potentiated inhibitor protein of 17kDa, called CPI-17, specifically inhibits myosin light chain phosphatase (MLCP). Phosphorylation of Thr-38 in vivo highly potentiates the ability of CPI-17 to inhibit MLCP. Thr-38 has been shown to be phosphorylated in vitro by a number of protein kinases including protein kinase C (PKC), Rho-associated coiled-coil kinase (ROCK), and protein kinase N (PKN). In this study we have focused on the association of protein kinases with CPI-17. Using affinity chromatography and Western blot analysis, we found interaction with all PKC isotypes and casein kinase I isoforms, CKIalpha and CKI. By contrast, ROCK and PKN did not associate with CPI-17, suggesting that PKC may be the relevant kinase that phosphorylates Thr-38 in vivo. CPI-17 interacted with the cysteine-rich domain of PKC and was phosphorylated by all PKC isotypes. We previously found that CPI-17 co-purified with casein kinase I in brain suggesting they are part of a complex and we now show that CPI-17 associates with the kinase domain of CKI isoforms.     

Defining the structural determinants and a potential mechanism for inhibition of myosin phosphatase by the protein kinase C-potentiated inhibitor protein of 17 kDa

Contractility of smooth muscle and non-muscle microfilaments involves phosphorylation of myosin II light chain. Myosin light chain phosphatase (MLCP) is specifically inhibited by the protein kinase C-potentiated inhibitor protein of 17 kDa, called CPI-17, as part of Ca(2+) sensitization of vascular smooth muscle contraction. Phosphorylation of Thr(38) in CPI-17 enhances inhibitory potency toward MLCP over 1000-fold. In this study we mapped regions of CPI-17 required for inhibition and investigated the mechanism using deletion and point mutants. Deletion of either the N-terminal 34 residues or C-terminal 27 residues gave no change in the IC(50) of either phospho- or unphospho-CPI-17. However, further deletion to give CPI-17 proteins of 1-102, 1-89, 1-76, and 1-67, resulted in much higher IC(50) values. The results indicate there is a minimal inhibitory domain between residues 35 and 120. A single Ala substitution at Tyr(41) eliminated phosphorylation-dependent inhibition, and phospho-Thr(38) in the Y41A protein was efficiently dephosphorylated by MLCP itself. The wild type CPI-17 expressed in fibroblast-induced bundling and contraction of actomyosin filaments, whereas expression of the Y41A protein had no obvious effects. Thus, a central domain of CPI-17(35-120) including phospho-Thr(38) is necessary for recognition by myosin phosphatase and Tyr(41) arrests dephosphorylation, thereby producing inhibition.     

Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility

Myosin light chain phosphatase (MLCP) plays a pivotal role in smooth muscle contraction by regulating Ca(2+) sensitivity of myosin light chain phosphorylation. A smooth muscle phosphoprotein called CPI-17 specifically and potently inhibits MLCP in vitro and in situ and is activated when phosphorylated at Thr-38, which increases its inhibitory potency 1000-fold. We produced a phosphospecific antibody for this site in CPI-17 and used it to study in situ phosphorylation of endogenous CPI-17 in arterial smooth muscle in response to agonist stimulation. In the intact femoral artery, CPI-17 phosphorylation was negligible at the resting state and was not increased during contraction induced by K(+) depolarization. The Ca(2+)-sensitizing agonists histamine and phenylephrine induced nearly equivalent contractions, but histamine generated significantly higher levels of CPI-17 phosphorylation. In alpha-toxin-permeabilized strips at pCa 6.7, contractile force and CPI-17 phosphorylation were proportional in response to histamine, guanosine 5'-O-(gamma-thiotriphosphate), and histamine plus guanyl-5'-yl thiophosphate, implying that histamine increased CPI-17 phosphorylation through activation of G proteins. Inhibitors of Rho-kinase (Y27632) and protein kinase C (PKC; GF109203X) reduced contraction and CPI-17 phosphorylation in parallel, suggesting that CPI-17 functions downstream of Rho kinases and PKC. The results show that agonists such as histamine signal through phosphorylation of CPI-17 to produce Ca(2+) sensitization of smooth muscle contraction.     

Phosphorylation of maize RAB-17 protein by casein kinase 2

The maize gene RAB-17, which is responsive to abscisic acid, encodes a basic glycine-rich protein containing, in the middle part of its sequence, a cluster of 8 serine residues followed by a putative casein kinase 2-type substrate consensus sequence. This protein was found to be highly phosphorylated in vivo. Here, we show that RAB-17 protein is a real substrate for casein kinase 2. RAB-17 protein is phosphorylated in vitro by casein kinase 2 isolated from rat liver cytosol and from maize embryos. A maximum of 4 mol of phosphate were incorporated per mol of RAB-17 protein following incubation with casein kinase 2. Phosphopeptide mapping experiments show that the peptide phosphorylated by casein kinase 2 in vitro is identical to that derived from the protein phosphorylated in vivo. Purification by high performance liquid chromatography and partial sequencing of the phosphopeptide indicate that it corresponds to the region of the protein (residues 56-89) containing the cluster of serine residues. Our results indicate that RAB-17 is phosphorylated by casein kinase 2 or a kinase with a similar specificity and that phosphorylation takes place in the serine cluster region of the protein both in vitro and in vivo.     

Phosphorylation of smooth muscle myosin light chain kinase by Ca2+-activated, phospholipid-dependent protein kinase

Protein kinase C incorporates phosphate into two sites of myosin light chain kinase (MLC-kinase) in the absence of calmodulin. Phosphorylation is all but abolished in the presence of Ca2+ and calmodulin, suggesting that both sites of phosphorylation are close to the calmodulin binding site. The phosphorylation of MLC-kinase results in an approximately 10-fold increase in the dissociation constant of MLC-kinase for calmodulin. Following phosphorylation (2 mol/mol of enzyme) of MLC-kinase by protein kinase C, an additional 2 mol of phosphate can be incorporated into the MLC-kinase apoenzyme by the cAMP-dependent protein kinase. Different maps of phosphopeptides were obtained by tryptic hydrolysis from MLC-kinase preparations phosphorylated by each kinase. The phosphorylation sites for the cAMP-dependent kinase were located in a fragment of approximately 25,000 daltons. In contrast the phosphorylation sites for protein kinase C are found in a much smaller tryptic peptide. These results suggest that the phosphorylation sites on MLC-kinase are different for protein kinase C and for cAMP-dependent protein kinase. However, phosphorylation in both regions results in a reduced affinity for calmodulin.     

Phosphorylation of a putative myosin light chain inChara by calcium-dependent protein kinase

Summary Ca²⁺-dependent protein kinase (CDPK) has been proposed to mediate inhibition by Ca²⁺ of cytoplasmic streaming in the green algaChara. We have identified the in vivo substrate(s) of CDPK inChara by using vacuolar perfusion of individual internodal cells with [-³²P]ATP. Phosphorylation of several polypeptides is enhanced when perfusions are performed at 10^–4M free Ca²⁺ compared to <10^–9M free Ca²⁺. The Ca²⁺-stimulated phosphorylation of these proteins is inhibited by the presence of a monoclonal antibody to soybean CDPK. One of these proteins is 16 to 18kDa and is recognized by an antibody against gizzard myosin light chains. These results demonstrate that inChara, several polypeptides are phophorylated by CDPK and one of these proteins has been tentatively identified as a myosin light chain. These observations support the hypothesis that Ca²⁺-regulated phosphorylation of myosin is involved in the regulation of cytoplasmic streaming.Abbreviations CDPK calcium-dependent protein kinase - mAb monoclonal antibody     

Histamine-induced vasoconstriction involves phosphorylation of a specific inhibitor protein for myosin phosphatase by protein kinase C alpha and delta isoforms

Histamine stimulus triggers inhibition of myosin phosphatase-enhanced phosphorylation of myosin and contraction of vascular smooth muscle. In response to histamine stimulation of intact femoral artery, a smooth muscle-specific protein called CPI-17 (for protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa) is phosphorylated and converted to a potent inhibitor for myosin phosphatase. Phosphorylation of CPI-17 is diminished by pretreatment with either or GF109203x, suggesting involvement of multiple kinases (Kitazawa, T., Eto, M., Woodsome, T. P., and Brautigan, D. L. (2000) J. Biol. Chem. 275, 9897--9900). Here we purified and identified CPI-17 kinases endogenous to pig artery that phosphorylate CPI-17. DEAE-Toyopearl column chromatography of aorta extracts separated two CPI-17 kinases. One kinase was protein kinase C (PKC) alpha, and the second kinase was purified to homogeneity as a 45-kDa protein, and identified by sequencing as PKC delta. Purified PKC delta was 3-fold more reactive with CPI-17 compared with myelin basic protein, whereas purified PKC alpha and recombinant RhoA-activated kinases (Rho-associated coiled-coil forming protein Ser/Thr kinase and protein kinase N) showed equal activity with CPI-17 and myelin basic protein. inhibited CPI-17 phosphorylation by purified PKC delta with IC(50) of 0.6 microm (in the presence of 0.1 mm ATP) or 14 microm (2.0 mm ATP). significantly suppressed CPI-17 phosphorylation in smooth muscle cells, and the contraction of permeabilized rabbit femoral artery induced by stimulation with phorbol ester. GF109203x inhibited phorbol ester-induced contraction of rabbit femoral artery by 80%, whereas a PKC alpha/beta inhibitor, Go6976, reduced contraction by 47%. The results imply that histamine stimulation elicits contraction of vascular smooth muscle through activation of PKC alpha and especially PKC delta to phosphorylate CPI-17.     

Myotonic dystrophy protein kinase phosphorylates the myosin phosphatase targeting subunit and inhibits myosin phosphatase activity

Myotonic dystrophy protein kinase (DMPK) and Rho-kinase are related. An important function of Rho-kinase is to phosphorylate the myosin-binding subunit of myosin phosphatase (MYPT1) and inhibit phosphatase activity. Experiments were carried out to determine if DMPK could function similarly. MYPT1 was phosphorylated by DMPK. The phosphorylation site(s) was in the C-terminal part of the molecule. DMPK was not inhibited by the Rho-kinase inhibitors, Y-27632 and HA-1077. Several approaches were taken to determine that a major site of phosphorylation was T654. Phosphorylation at T654 inhibited phosphatase activity. Thus both DMPK and Rho-kinase may regulate myosin II phosphorylation.     

Distinctive solution conformation of phosphatase inhibitor CPI-17 substituted with aspartate at the phosphorylation-site threonine residue

We present solution NMR structures for wild-type and mutated forms of CPI-17, a phosphoinhibitor for protein phosphatase 1. Phosphorylation of Thr38 of CPI-17 produces a >1000-fold increase in inhibitory potency for myosin phosphatase. We compared the 1H-15N heteronuclear single quantum coherence spectroscopy (HSQC) chemical shifts of wild-type CPI-17, partially phosphorylated CPI-17 and CPI-17 with Thr38 replaced with Asp to introduce a negative charge. There was a switch in the protein conformation due to either Asp substitution or phosphorylation, so we determined the solution NMR structure of the CPI-17 T38D mutant as a model for the active (phospho-) conformation. The structures reveal a molecular switch in conformation that involves the rotation of two of the four helices in the four helix bundle. Despite this conformational switch, there was little increase in the inhibitory potency with T38D. We propose that for this inhibitor, a negative charge at residue 38 is sufficient to trigger an active conformation, but a phosphoryl group is required for full inhibitory potency against protein phosphatase-1.     

Effects of the phosphorylation of myosin phosphatase by cyclic GMP-dependent protein kinase.

Cyclic GMP-dependent protein kinase (PKG) phosphorylated, in vitro, the large (MYPT1) and small (M20) regulatory subunits of myosin phosphatase (MP) with maximum stoichiometries of 1.8 and 0.6 mol of phosphate/mol subunit, respectively. The phosphorylation of these subunits by PKG did not affect the phosphatase activity towards the 20 kDa myosin light chain. However, phosphorylation of the MP holoenzyme decreased the binding of MP to phospholipid. The phosphorylation of the serine residue of the C-terminal part of MYPT1 was crucial for these interactions. These results suggest that the phosphorylation of MP by PKG is not a direct mechanism in activating MP activity, and that other indirect mechanisms, including the interaction between MP and phospholipids, might be candidates for Ca2+ desensitization via cGMP in smooth muscle.     

Phosphorylation of smooth muscle myosin by type II Ca2+/calmodulin-dependent protein kinase

Brain type II Ca²⁺/calmodulin-dependent protein kinase was found to phoshorylate smooth muscle myosin, incorporating maximally 2 mol of phosphoryl per mol of myosin, exclusively on the 20,000 dalton light chain subunit. After maximal phosphorylation of myosin or the isolated 20,000 dalton light chain subunit by myosin light chain kinase, the addition of type II Ca²⁺/calmodulin-dependent protein kinase led to no further incorporation indicating the two kinases phosphorylated a common site. This conclusion was supported by two dimensional mapping of tryptic digests of myosin phosphorylated by the two kinases. By phosphoamino acid analysis the phosphorylated residue was identified as a serine. The phosphorylation by type II Ca ²⁺/calmodulin-dependent protein kinase of myosin resulted in enhancement of its actin-activated Mg²⁺-ATPase activity. Taken together, these data strongly support the conclusion that type II Ca²⁺/calmodulin-dependent protein kinase phosphorylates the same amino acid residue on the 20,000 dalton light chain subunit of smooth muscle myosin as is phosphorylated by myosin light chain kinase and suggest an alternative mechanism for the regulation of actin-myosin interaction.Abbreviations SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - EGTA Ethylene Glycol Bis (-amino-ethyl ether)-N,N,N,N-Tetraacetic Acid - DTT Dithiothreitol - LC₂₀ Gizzard Smooth Muscle Phosphorylatable 20 kDa Myosin Light Chain - LC₁₇ Gizzard Smooth Muscle, 17 kDa Myosin Light Chain - H Chain Gizzard Smooth Muscle 200 kDa Myosin Heavy Chain - TPCK L-1-Tosylamido-2-Phenylethyl Chloromethyl Ketone - MOPS 3-(N-morpholino) Propanesulfonic Acid