Protein tyrosine phosphorylation smooth muscle activation

Molloy, C. J., Taylor, D. S., and Weber, H. 1993. Angiotensin II stimulation of rapid protein tyrosine phosphorylation and protein kinase activation in rat aortic smooth muscle cells. J Biol Chem 268 7338-7345. 

In this section we review studies performed in our laboratory that strongly suggest that tyrosine kinase activity and protein tyrosine phosphorylation are important mechanisms for regulating receptor-activated increases in [Ca +Jj in smooth muscle. 

Accordingly, the maintenance of tyrosine phosphorylation during relaxation induced by chelation of extracellular Ca + with EGTA probably reflects persistent inhibition of tyrosine phosphatase activity by the continued presence of vanadate. The tyrosine-phos-phorylated substrates may be poised to promote Ca + entry so that replacement of the medium with fresh Ca2+-PSS allows for rapid entry of Ca2+ and a large spontaneous transient contraction. In contrast, no spontaneous contraction occurred in the experiment with La3+ and vanadate because La3+ blockade of the Ca + channels persists when the medium is replaced with fresh Ca2+-PSS (Fig. 2D). Based on this interpretation of the data, further studies were performed to more directly test the hypothesis that protein tyrosine phosphorylation is an important mechanism for regulating [Ca +Jj in smooth muscle cells. 

C. Smooth Muscle Activation and Enhanced Protein Tyrosine Phosphorylation tasGAP Is a Substrate... 

Di Salvo J Nelson SR, Kaplan N (1997) Protein tyrosine phosphorylation in smooth muscle a potential coupling mechanism between receptor activation and intracellular calcium. Proc Soc Bxp Biol Med 214 285-301 Erdodi F, Ito M, Hartshome DJ. (1996) Myosin light chain phosphatase. In Bdrany M (ed) Biochemistry of smooth muscle contraction. Academic Press, San Diego, pp 13M42... 

FIGU R E 7 A similar set of substrates is tyrosine phosphorylated during activation of either intact taenia coli, cultured VSMC, or staphylococcal a-toxin-permeabilized deal longitudinal smooth muscle. In these experiments, tyrosine.-phosphorylated substrates were detected by immunoblotting with antiphosphotyrosine antibodies and enhanced chemiluminescence technology rather than the less sensitive I-labeled protein A technology used in Fig. 2. Stimulation of guinea pig taenia coli with either 10 jcM carbachol (Carb) or 1.5 mM vanadate (Van) resulted in pronounced tyrosine phosphorylation of at least nine substrates with apparent masses of 42-45, 50, 70, 80-85, 95,100, 110, 116, and 205 kDa. In like fashion, stimulation of canine femoral VSMC with 100 jjlM phenylephrine (PE) resulted in enhanced tyrosine phosphorylation of a similar set of substrates (however, note that qualitative differences were evident with respect to some substrates, such as the one of 205 kDa). Similarly, the same substrates appeared to be tyrosine phosphorylated when permeabilized ileal smooth muscle was contracted with Ca + (pCa 4.5). From Di Salvo et al. (1994), Fig. 5, p. 1438. 

Other evidence has shown significant tyrosine kinase and MAP kinase activities in adult, terminally differentiated smooth muscle cells (Di Salvo et al., 1989 Adam et ah, 1992 Childs et al., 1992) upon their stimulation with nonmitogenic agonists. MAP kinase transiently translocates to the surface membrane during early activation, but during maintained activation a second redistribution of MAP kinase from the surface membrane to the cytoskeleton is observed (Khalil and Morgan, 1993). The initial association of MAP kinase appears to require upstream PKC activity but occurs in a tyrosine phosphorylation-independent manner. In contrast, the delayed redistribution of MAP kinase occurs in a tyrosine phosphorylation-dependent manner and appears to target MAP kinase to the contractile proteins (Khalil et al., 1995). 

The release of InsPa from phosphatidylinositol 4,5 bisphosphate (PIP2) is initiated by the binding of excitatory agonists to heptameric serpentine receptors coupled to trimeric G-proteins (GOq, Gan) that activate PLCp (smooth muscle, see LaBelle and Polyak 1996) or through (tyrosine) phosphorylation of PLCy. InsPa-induced Ca +-release requires adenosine nucleotide (Smith et al. 1985 Somlyo et al. 1992) and can be modulated by [Ca " "] (lino 1987 lino and Endo 1992). 

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