It is clear from several studies that myosin phosphatase (MP) can be inhibited via a pathway that involves RhoA. However, the mechanism of inhibition is not established. These studies were carried out to test the hypothesis that Rho-kinase (Rho-associated kinase) via phosphorylation of the myosin phosphatase target subunit 1 (MYPT1) inhibited MP activity and to identify relevant sites of phosphorylation. Phosphorylation by Rho-kinase inhibited MP activity and this reflected a decrease in V max . Activity of MP with different substrates also was inhibited by phosphorylation.

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Protein phosphorylation may play a critical role in stimulus-coupled secretion of platelets. Some platelet proteins become phosphorylated on exposure to agents such as thrombin and collagen, and the smallest of these phosphoproteins (molecular weight 20,000), has been identified as a light chain of myosin. Phosphorylation of myosin light chain increases the activity of actin-activated myosin ATPase and the resultant contraction of the actomyosin presumably mediates the release reaction. Platelet myosin light chain kinase has been identified as a calcium-dependent enzyme requiring calmodulin for its activity. Calmodulin is a Ca2+-binding protein with a molecular weight of approximately 18,000 which seems to be involved in a wide variety of cellular processes. Although a growing body of evidence suggests that non-muscle actomyosin, such as that isolated from platelets, is regulated by Ca2+-calmodulin-dependent light chain phosphorylation, the precise relationship between the phosphorylation and the function of platelets is not clearly established. We now present pharmacological evidence that a calmodulin-mediated system, such as Ca2+-dependent myosin light chain phosphorylation, also plays an important role in the phenomenon of the release reaction. N-(6-aminohexyl)-5-chloro-1-napthalene-sulphonamide (W-7) (refs 13-15) is shown to bind selectively to calmodulin in vitro and inhibit its biological activity.

Phosphorylation of the 20,000 molecular weight (MW) light chain of platelet myosin is associated with the activation of platelets and subsequent release of platelet granules, and the protein kinase catalysing this phosphorylation has been identified as the Ca2+/calmodulin-dependent enzyme, myosin light chain kinase. Tumour-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA), which activate Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), can also cause platelet aggregation and phosphorylation of a 20,000-MW peptide in blood platelets. It was therefore of interest to ascertain whether the 20,000-MW peptide phosphorylated in platelets was the light chain of myosin and whether TPA-induced phosphorylation of the 20,000-MW peptide could be differentiated from thrombin-induced phosphorylation. We now report that TPA-induced activation of platelets is associated with the phosphorylation of the 20,000-MW light chain of myosin, that it appears to be mediated mainly through protein kinase C and that the site phosphorylated in the myosin light chain is distinct from that phosphorylated by myosin light chain kinase.

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Purpose: We developed a complex of tumor antigen protein with a novel nanoparticle antigen delivery system of cholesteryl pullulan (CHP). To target HER2 antigen, we prepared truncated HER2 protein 1-146 (146HER2) complexed with CHP, the CHP-HER2 vaccine. We designed a clinical study to assess the safety of the vaccine and HER2-specific T-cell immune responses measured by the newly developed enzyme-linked immunospot assay with mRNA-transduced phytohemagglutinin-stimulated CD4 + T cells in HLA-A2402-positive patients with therapyrefractory HER2-expressing cancers. Experimental Design: Nine patients with various types of solid tumors were enrolled. Each patient was s.c. vaccinated biweekly with 300 Ag of CHP-HER2 vaccine for three times followed by booster doses. HER2-specific T-cell responses were evaluated by enzyme-linked immunospot assay by targeting autologous phytohemagglutinin-stimulated CD4 + T cells transduced with 146HER2-encoding mRNA to cover both identified peptides and unknown epitopes for MHC class I and class II that might exist in the sequence of the vaccine protein.Results: CHP-HER2 vaccine was well tolerated; the only adverse effect was grade1transient skin reaction at the sites of vaccination. HER2-specific CD8 + and/or CD4 + T-cell immune responses were detected in five patients who received four to eight vaccinations, among whom bothT-cell responses were detected in these patients. In four patients with CD8 + T-cell responses, two patients reacted to previously identified HER2 63-71 peptide and the other two reacted only to 146HER2 mRNA-transduced cells. Conclusions: CHP-HER2 vaccine was safe and induced HER2-specific CD8 + and/or CD4 + T-cell immune responses.

Protein kinase C (PKC)–potentiated inhibitory phosphoprotein of myosin phosphatase (CPI) was detected in human platelets. Like smooth muscle CPI-17, in vitro phosphorylation of platelet CPI by PKC inhibited the activity of myosin phosphatase containing the PP1δ catalytic subunit and the 130-kd myosin-binding subunit (MBS). Treatment of intact platelets with thrombin or the stable thromboxane A2 analog STA2 resulted in increased phosphorylation of both CPI and MBS at Thr-696, whereas phorbol myristate acetate (PMA) and the Ca++ ionophore ionomycin only induced CPI phosphorylation. PMA induced slow adenosine triphosphate (ATP) secretion of fura 2–loaded platelets with no change in cytosolic Ca++. The PMA-induced increase in CPI phosphorylation preceded phosphorylation of 20-kd myosin light chain (MLC20) at Ser-19 and ATP secretion. The PKC inhibitor, GF109203X, inhibited PMA-induced phosphorylation of CPI and MLC20 with similar IC50 values. These findings suggest that the activation of PKC by PMA induces MLC20phosphorylation by inhibiting myosin phosphatase through phosphorylation of CPI. STA2-induced MLC20phosphorylation was also diminished but not abolished by GF109203X, even at high concentrations that completely inhibited STA2-induced CPI phosphorylation. A combination of the Rho-kinase inhibitor Y-27632 and GF109203X led to a further decrease in STA2-induced MLC20 phosphorylation, mainly because of a significant inhibition of MBS phosphorylation at Thr-696. Inhibition of STA2-induced ATP release by Y-27632, GF109203X, or both appeared to correlate with the extent of MLC20 phosphorylation. Thus, CPI phosphorylation by PKC may participate in inhibiting myosin phosphatase, in addition to the Rho-kinase–mediated regulation of myosin phosphatase, during agonist-induced platelet secretion.

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