Myosin Phosphatase Target Subunit 1 (MYPT1) Regulates the Contraction and Relaxation of Vascular Smooth Muscle and Maintains Blood Pressure

Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca²⁺-dependent phosphorylation of myosin light chain by myosin light chain kinase, which is essential for smooth muscle contraction. The role of MYPT1 in vascular smooth muscle was investigated in adult MYPT1 smooth muscle specific knock-out mice. MYPT1 deletion enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries treated with KCl and various vascular agonists. The contractile responses of arteries from knock-out mice to norepinephrine were inhibited by Rho-associated kinase (ROCK) and protein kinase C inhibitors and were associated with inhibition of phosphorylation of the myosin light chain phosphatase inhibitor CPI-17. Additionally, stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway still resulted in relaxation of MYPT1-deficient mesenteric arteries, indicating phosphorylation of MYPT1 by PKG is not a major contributor to the relaxation response. Thus, MYPT1 enhances myosin light chain phosphatase activity sufficient for blood pressure maintenance. Rho-associated kinase phosphorylation of CPI-17 plays a significant role in enhancing vascular contractile responses, whereas phosphorylation of MYPT1 in the NO/cGMP/PKG signaling module is not necessary for relaxation.     

Phosphorylation is required for myosin regulatory light chain (PmMRLC) to control yellow head virus infection in shrimp hemocytes

The cellular signal-transduction process is largely controlled by protein phosphorylation. Shrimp infected with yellow head virus show dramatic changes in their hemocyte phosphoproteomic patterns, and aberrant activation of phosphorylation-based signaling networks has been implicated in a number of diseases. In this study, we focused on phosphorylation of Penaeus monodon myosin regulatory light chain (PmMRLC) that is induced at an early hour post YHV infection and is concomitant with cellular actin remodeling. In shrimp cell cultures, this phosphorylation was inhibited by the myosin light chain kinase (MLCK) inhibitors ML-7 and ML-9, suggesting that PmMLC phosphorylation is MLCK pathway-dependent. Blocking PmMRLC phosphorylation resulted in increased replication of YHV and reduction of phagocytic activities of shrimp hemocytes called semigranular cells (SGC) and granular cells (GC). Injection of MLCK inhibitors prior to YHV challenge resulted in dose-dependent elevation in quantity of YHV-positive GC and cytoplasmic YHV protein, coincident with high shrimp mortality. Altogether, we demonstrated that PmMRLC phosphorylation increases after YHV infection in shrimp and that inhibition of the phosphorylation leads to increased YHV replication, reduced hemocyte phagocytic activity (probably through actin remodeling) and subsequent shrimp death. Thus, further studies on the MLCK activation pathway may lead to new strategies in development and implementation of therapy for YHV infections in shrimp.     

Light activation of maize phosphoenolpyruvate carboxylase protein-serine kinase activity is inhibited by mesophyll and bundle sheath-directed photosynthesis inhibitors

C₄ phosphoenolpyruvate carboxylase (PEPC) is post-translationally regulated by reversible phosphorylation of a specific N-terminal seryl residue in response to light/dark transitions of the parent leaf tissue. The protein-serine kinase (PEPC-PK) that phosphorylates/activates this mesophyll-cytoplasm target enzyme is slowly, but strikingly, activated by high light and inactivated in darkness in vivo by a mechanism involving cytoplasmic protein synthesis/degradation as a primary component. In this report, evidence is presented indicating that the inhibition of Calvin cycle activity by a variety of mesophyll (3-(3,4-dichlorophenyl)-1,1-dimethylurea, isocil, methyl viologen) and bundle sheath (dl-glyceraldehyde)-directed photosynthesis inhibitors blocks the light activation of maize (Zea mays L.) PEPC-PK and the ensuing regulatory phosphorylation of its target enzyme in vivo. Based on these and related observations, we propose that the Calvin cycle supplies the C₄ mesophyll cell with (a) a putative signal (e.g. phosphorylated metabolite, amino acid) that interacts with the cytoplasmic protein synthesis event to effect the light activation of PEPC-PK and the concomitant phosphorylation of PEPC, and (b) high levels of known positive effectors (e.g. triose-phosphate, glucose-6-phosphate) that interact directly with the carboxylase. The combined result of this complex regulatory cascade is to effectively desensitize PEPC to feedback inhibition by the millimolar levels of l-malate required for rapid diffusive transport to the bundle sheath during high rates of C₄ photosynthesis.     

Photomorphogenesis in<Emphasis Type="Italic"> Phycomyces</Emphasis>: differential display of gene expression by PCR with arbitrary primers

The zygomycete fungus Phycomyces blakesleeanus develops two types of fruiting bodies of very different size, macrophores and microphores. Blue light stimulates macrophorogenesis and inhibits microphorogenesis. I have adapted a method based on the polymerase chain reaction with arbitrary primers to investigate the role of differential gene expression during photophorogenesis in Phycomyces. Several cDNAs for genes induced in vegetative mycelium have been observed, but only one gene induced by blue light has been detected. I have demonstrated the feasibility of this approach by the isolation of a cDNA segment for the heat-shock protein HSP100 that is induced by blue light at the onset of sporangiophore development. The heat-shock protein HSP100 is an ATP-binding protein that has the ability to disassemble protein complexes. In plants, the gene for HSP100 is induced by light. The cDNA segment for HSP100 obtained from Phycomyces is 686 bp long and the predicted amino acid sequence contains one of the ATP-binding sites.     

Staurosporine inhibition of zipper-interacting protein kinase contractile effects in gastrointestinal smooth muscle.

Zipper-interacting protein kinase (ZIPK) is a serine-threonine kinase that has been implicated in Ca2+-independent myosin II phosphorylation and contractile force generation in vascular smooth muscle. However, relatively little is known about the contribution of this kinase to gastrointestinal smooth muscle contraction. The addition of a recombinant version of ZIPK that lacked the leucine zipper domain to permeabilized ileal strips evoked a Ca2+-independent contraction and resulted in myosin regulatory light chain diphosphorylation at Ser19 and Thr18. Neither Ca2+-independent force development nor myosin regulatory light chain phosphorylation was elicited by the addition of kinase-dead ZIPK to the ileal strips. The sensitivity of ZIPK-induced contraction to various kinase inhibitors was similar to the in vitro sensitivity of purified ZIPK to these inhibitors. Staurosporine was the most effective ZIPK inhibitor, with a Ki value calculated to be 2.6 +/- 0.3 micromol/L. Through the use of specific kinase inhibitors, we determined that Rho-associated protein kinase and Ca2+/phospholipid-dependent protein kinase (protein kinase C) do not mitigate ZIPK-induced contraction in ileum. Our findings support a role for ZIPK in Ca2+-independent contractile force generation in gastrointestinal smooth muscle.     

Nonmuscle myosin II is responsible for maintaining endothelial cell basal tone and stress fiber integrity

Cultured confluent endothelial cells exhibit stable basal isometric tone associated with constitutive myosin II regulatory light chain (RLC) phosphorylation. Thrombin treatment causes a rapid increase in isometric tension concomitant with myosin II RLC phosphorylation, actin polymerization, and stress fiber reorganization while inhibitors of myosin light chain kinase (MLCK) and Rho-kinase prevent these responses. These findings suggest a central role for myosin II in the regulation of endothelial cell tension. The present studies examine the effects of blebbistatin, a specific inhibitor of myosin II activity, on basal tone and thrombin-induced tension development. Although blebbistatin treatment abolished basal tension, this was accompanied by an increase in myosin II RLC phosphorylation. The increase in RLC phosphorylation was Ca²⁺ dependent and mediated by MLCK. Similarly, blebbistatin inhibited thrombin-induced tension without interfering with the increase in RLC phosphorylation or in F-actin polymerization. Blebbistatin did prevent myosin II filament incorporation and association with polymerizing or reorganized actin filaments leading to the disappearance of stress fibers. Thus the inhibitory effects of blebbistatin on basal tone and induced tension are consistent with a requirement for myosin II activity to maintain stress fiber integrity. actin; blebbistatin; isometric tension; myosin light chain kinase; regulatory light chain phosphorylation; focal adhesions     

Pyruvate,orthophosphate dikinase in leaves and chloroplasts of C(3) plants undergoes light-/dark-induced reversible phosphorylation

Pyruvate,orthophosphate (Pi) dikinase (PPDK) is best recognized as a chloroplastic C(4) cycle enzyme. As one of the key regulatory foci for controlling flux through this photosynthetic pathway, it is strictly and reversibly regulated by light. This light/dark modulation is mediated by reversible phosphorylation of a conserved threonine residue in the active-site domain by the PPDK regulatory protein (RP), a bifunctional protein kinase/phosphatase. PPDK is also present in C(3) plants, although it has no known photosynthetic function. Nevertheless, in this report we show that C(3) PPDK in leaves of several angiosperms and in isolated intact spinach (Spinacia oleracea) chloroplasts undergoes light-/dark-induced changes in phosphorylation state in a manner similar to C(4) dikinase. In addition, the kinetics of this process closely resemble the reversible C(4) process, with light-induced dephosphorylation occurring rapidly (< or =15 min) and dark-induced phosphorylation occurring much more slowly (> or =30-60 min). In intact spinach chloroplasts, light-induced dephosphorylation of C(3) PPDK was shown to be dependent on exogenous Pi and photosystem II activity but independent of electron transfer from photosystem I. These in organello results implicate a role for stromal pools of Pi and adenylates in regulating the reversible phosphorylation of C(3)-PPDK. Last, we used an in vitro RP assay to directly demonstrate ADP-dependent PPDK phosphorylation in desalted leaf extracts of the C(3) plants Vicia faba and rice (Oryza sativa). We conclude that an RP-like activity mediates the light/dark modulation of PPDK phosphorylation state in C(3) leaves and chloroplasts and likely represents the ancestral isoform of this unusual and key C(4) pathway regulatory "converter" enzyme.     

The regulation of smooth muscle contractility by zipper-interacting protein kinase

Smooth muscle contractility is mainly regulated by phosphorylation of the 20 kDa myosin light chains (LC20), a process that is controlled by the opposing activities of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Recently, intensive research has revealed that various protein kinase networks including Rho-kinase, integrin-linked kinase, zipper-interacting protein kinase (ZIPK), and protein kinase C (PKC) are involved in the regulation of LC20 phosphorylation and have important roles in modulating smooth muscle contractile responses to Ca2+ (i.e., Ca2+ sensitization and Ca2+ desensitization). Here, we review the general background and structure of ZIPK and summarize our current understanding of its involvement in a number of cell processes including cell death (apoptosis), cell motility, and smooth muscle contraction. ZIPK has been found to induce the diphosphorylation of LC20 at Ser-19 and Thr-18 in a Ca2+-independent manner and to regulate MLCP activity directly through its phosphorylation of the myosin-targeting subunit of MLCP or indirectly through its phosphorylation of the PKC-potentiated inhibitory protein of MLCP. Future investigations of ZIPK function in smooth muscle will undoubtably focus on determining the mechanisms that regulate its cellular activity, including the identification of upstream signaling pathways, the characterization of autoinhibitory domains and regulatory phosphorylation sites, and the development of specific inhibitor compounds.     

Defining the substrate specificity determinants recognized by the active site of C-terminal Src kinase-homologous kinase (CHK) and identification of β-synuclein as a potential CHK physiological substrate

C-Terminal Src kinase-homologous kinase (CHK) exerts its tumor suppressor function by phosphorylating the C-terminal regulatory tyrosine of the Src-family kinases (SFKs). The phosphorylation suppresses their activity and oncogenic action. In addition to phosphorylating SFKs, CHK also performs non-SFK-related functions by phosphorylating other cellular protein substrates. To define these non-SFK-related functions of CHK, we used the "kinase substrate tracking and elucidation" method to search for its potential physiological substrates in rat brain cytosol. Our search revealed β-synuclein as a potential CHK substrate, and Y127 in β-synuclein as the preferential phosphorylation site. Using peptides derived from β-synuclein and positional scanning combinatorial peptide library screening, we defined the optimal substrate phosphorylation sequence recognized by the CHK active site to be E-x-[Φ/E/D]-Y-Φ-x-Φ, where Φ and x represent hydrophobic residues and any residue, respectively. Besides β-synuclein, cellular proteins containing motifs resembling this sequence are potential CHK substrates. Intriguingly, the CHK-optimal substrate phosphorylation sequence bears little resemblance to the C-terminal tail sequence of SFKs, indicating that interactions between the CHK active site and the local determinants near the C-terminal regulatory tyrosine of SFKs play only a minor role in governing specific phosphorylation of SFKs by CHK. Our results imply that recognition of SFKs by CHK is mainly governed by interactions between motifs located distally from the active site of CHK and determinants spatially separate from the C-terminal regulatory tyrosine in SFKs. Thus, besides assisting in the identification of potential CHK physiological substrates, our findings shed new light on how CHK recognizes SFKs and other protein substrates.     

Gibberellin-mediated proteasome-dependent degradation of the barley DELLA protein SLN1 repressor

DELLA proteins are nuclear repressors of plant gibberellin (GA) responses. Here, we investigate the properties of SLN1, a DELLA protein from barley that is destabilized by GA treatment. Using specific inhibitors of proteasome function, we show that proteasome-mediated protein degradation is necessary for GA-mediated destabilization of SLN1. We also show that GA responses, such as the aleurone alpha-amylase response and seedling leaf extension growth, require proteasome-dependent GA-mediated SLN1 destabilization. In further experiments with protein kinase and protein phosphatase inhibitors, we identify two additional signaling steps that are necessary for GA response and for GA-mediated destabilization of SLN1. Thus, GA signaling involves protein phosphorylation and dephosphorylation steps and promotes the derepression of GA responses via proteasome-dependent destabilization of DELLA repressors.     

Regulation of myosin-bound protein phosphatase by insulin in vascular smooth muscle cells: evaluation of the role of Rho kinase and phosphatidylinositol-3-kinase-dependent signaling pathways

In this study, we examined the molecular mechanism of myosin-bound protein phosphatase (MBP) regulation by insulin and evaluated the role of MBP in insulin-mediated vasorelaxation. Insulin rapidly stimulated MBP in confluent primary vascular smooth muscle cell (VSMC) cultures. In contrast, VSMCs isolated from diabetic and hypertensive rats exhibited impaired MBP activation by insulin. Insulin-mediated MBP activation was accompanied by a rapid time-dependent reduction in the phosphorylation state of the myosin-bound regulatory subunit (MBS) of MBP. The decrease observed in MBS phosphorylation was due to insulin-induced inhibition of Rho kinase activity. Insulin also prevented a thrombin-mediated increase in Rho kinase activation and abolished the thrombin-induced increase in MBS phosphorylation and MBP inactivation. These data are consistent with the notion that insulin inactivates Rho kinase and decreases MBS phosphorylation to activate MBP in VSMCs. Furthermore, treatment with synthetic inhibitors of phosphatidylinositol-3 kinase (PI3-kinase), nitric oxide synthase (NOS), and cyclic guanosine monophosphate (cGMP) all blocked insulin's effect on MBP activation. We conclude that insulin stimulates MBP via its regulatory subunit, MBS partly by inactivating Rho kinase and stimulating NO/cGMP signaling via PI3-kinase as part of a complex signaling network that controls 20-kDa myosin light chain (MLC20) phosphorylation and VSMC contraction.     

Cyclosporine-induced renal artery smooth muscle contraction is associated with increases in the phosphorylation of specific contractile regulatory proteins

Cyclosporine A (CSA) is a type 2B phosphatase inhibitor which can induce contraction of renal artery smooth muscle. In this investigation, we examined the phosphorylation events associated with CSA-induced contraction of bovine renal artery smooth muscle. Contractile responses were determined in a muscle bath and the corresponding phosphorylation events were determined with whole cell phosphorylation and two-dimensional gel electrophoresis. CSA-induced contractions were associated with increases in the phosphorylation of the 20 kDa myosin light chains (MLC20) and different isoforms of the small heat shock protein, HSP27. Cyclic nucleotide-dependent relaxation of CSA-induced contractions was associated with increases in the phosphorylation of another small heat shock protein, HSP20, and decreases in the phosphorylation of the MLC20, and some isoforms of HSP27. These data suggest that CSA-induced contraction and relaxation of vascular smooth muscle is associated with increases in the phosphorylation of specific contractile regulatory proteins.     

Signaling through Myosin Light Chain Kinase in Smooth Muscles

Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates smooth muscle myosin regulatory light chain (RLC) to initiate contraction. We used a tamoxifen-activated, smooth muscle-specific inactivation of MLCK expression in adult mice to determine whether MLCK was differentially limiting in distinct smooth muscles. A 50% decrease in MLCK in urinary bladder smooth muscle had no effect on RLC phosphorylation or on contractile responses, whereas an 80% decrease resulted in only a 20% decrease in RLC phosphorylation and contractile responses to the muscarinic agonist carbachol. Phosphorylation of the myosin light chain phosphatase regulatory subunit MYPT1 at Thr-696 and Thr-853 and the inhibitor protein CPI-17 were also stimulated with carbachol. These results are consistent with the previous findings that activation of a small fraction of MLCK by limiting amounts of free Ca²⁺/calmodulin combined with myosin light chain phosphatase inhibition is sufficient for robust RLC phosphorylation and contractile responses in bladder smooth muscle. In contrast, a 50% decrease in MLCK in aortic smooth muscle resulted in 40% inhibition of RLC phosphorylation and aorta contractile responses, whereas a 90% decrease profoundly inhibited both responses. Thus, MLCK content is limiting for contraction in aortic smooth muscle. Phosphorylation of CPI-17 and MYPT1 at Thr-696 and Thr-853 were also stimulated with phenylephrine but significantly less than in bladder tissue. These results indicate differential contributions of MLCK to signaling. Limiting MLCK activity combined with modest Ca²⁺ sensitization responses provide insights into how haploinsufficiency of MLCK may result in contractile dysfunction in vivo, leading to dissections of human thoracic aorta.     

Targeting the mTOR signaling network in cancer

The mammalian target of rapamycin (mTOR) is an unconventional protein kinase that is centrally involved in the control of cancer cell metabolism, growth and proliferation. The mTOR pathway has attracted broad scientific and clinical interest, particularly in light of the ongoing clinical cancer trials with mTOR inhibitors. The mixed clinical results to date reflect the complexity of both cancer as a disease target, and the mTOR signaling network, which contains two functionally distinct mTOR complexes, parallel regulatory pathways, and feedback loops that contribute to the variable cellular responses to the current inhibitors. In this review, we discuss the regulatory pathways that govern mTOR activity, and highlight clinical results obtained with the first generation of mTOR inhibitors to reach the oncology clinics.     

Substrate based inhibitors of smooth muscle myosin light chain kinase.

Activation of myosin light chain kinase is a prerequisite for smooth muscle activation. In this study, short peptide analogs of the phosphorylation site of the myosin light chain were studied for their effects on several contractile protein systems. The peptides inhibited phosphorylation of isolated ventricular and smooth muscle myosin light chains by smooth muscle myosin light chain kinase, but they were only weak inhibitors of phosphorylation of intact myosin and actomyosin. The peptides were also unable to block force development or myosin light chain phosphorylation in glycerol permeabilized fibers of swine carotid media. Apparently, the association of the myosin light chain with myosin changes its conformation such that substrate analogs which are potent inhibitors of the phosphorylation of isolated myosin light chains by myosin light chain kinase are ineffective at blocking phosphorylation of the intact molecule.     

siRNA knock down of casein kinase 2 increases force and cross-bridge cycling rates in vascular smooth muscle

Contraction of smooth muscle involves myosin light chain (MLC) kinase catalyzed phosphorylation of the regulatory MLC, activation of myosin, and the development of force. However, this cannot account for all aspects of a smooth muscle contraction, suggesting that other regulatory mechanisms exist. One potentially important technique to study alternative sites of contractile regulation is the use of small interfering RNA (siRNA). The goal of this study was to determine whether siRNA technology can decrease the levels of a specific protein and allow for the determination of how that protein affects contractile regulation. To achieve this goal, we tested the hypothesis that casein kinase 2 (CK2) is part of the complex regulatory scheme present in vascular smooth muscle. Using intact strips of swine carotid artery, we determined that siRNA against CK2 produced a tissue that resulted in a 60% knockdown after 4 days in organ culture. Intact strips of vascular tissue depleted of CK2 produced greater levels of force and exhibited an increased sensitivity to all stimuli tested. This was accompanied by an increase in cross-bridge cycling rates but not by a change in MLC phosphorylation levels. -Toxin-permeabilized vascular tissue depleted of CK2 also showed an increased sensitivity to calcium compared with control tissues. Our results demonstrate that siRNA is a viable technique with which to study regulatory pathways in intact smooth muscle tissue. Our results also demonstrate that CK2 plays an important role in the mechanism(s) responsible for the development of force and cross-bridge cycling by a MLC phosphorylation-independent pathway. myosin light chain phosphorylation; shortening velocity; -toxin permeabilization; swine carotid artery; caldesmon     

Abl functions as a negative regulator of Met-induced cell motility via phosphorylation of the adapter protein CrkII

HGF, the ligand for the Met receptor tyrosine kinase, is a potent modulator of epithelial-mesenchymal transition and dispersal of epithelial cells, which are processes that play a crucial role in cell motility during normal development and malignant transformation. We and others have shown earlier that the adapter protein CrkII and its associated proteins positively regulate cell migratory events in response to both haptotactic and chemotactic stimuli, including HGF. Here, we demonstrate for the first time that phosphorylation of CrkII serves as a negative feedback loop to regulate motile responses upon Met stimulation. Thus, we found that the treatment of cells with HGF induces tyrosine phosphorylation of CrkII at Y221, which in turn results in inhibition of CrkII signaling via formation of an intramolecular pY221-SH2-domain interaction. Accordingly, expression of a mutant form of CrkII, CrkII-Y221F, which is resistant to phosphorylation at this negative regulatory site, enhanced Met-induced cell motility. Furthermore, we demonstrate here that the Met-induced CrkII phosphorylation depends on the Abl tyrosine kinase activity. As a corollary, we found that Abl inhibitors, such as the STI571 compound, significantly enhanced Met-induced cell motility, but failed to do so in cells that expressed the CrkII-Y221F mutant protein. Taken together, these results demonstrate that the Abl tyrosine kinase functions as a negative regulator of Met-induced cell migration, and that it does so by inducing CrkII phosphorylation at the site Y221.