Small GTP binding proteins transduce signals that control a host of cellular responses. The activation of protein kinases by the GTP-bound form of small G proteins and highly regulated changes in the actin cytoskeleton appear to be important characteristics of the signalling properties of specific GTPases. Myosin II is an actin based molecular motor that converts chemical energy into mechanical work. The actin- myosin II interaction in smooth muscles is regulated by the phosphorylation of ser 19 on the 20 kDa light chain of smooth muscle myosin by the calcium-calmodulin dependent enzyme myosin light chain kinase. Recent reports have suggested that GTPases and myosin phosphorylation interact to regulate the actin cytoskeleton and smooth muscle contraction. Experiments performed by Somlyo, Kaibuchi and Narumiya and their co-workers have shown that GTPases regulate the calcium sensitivity of smooth muscle contraction. We have recently discovered that myosin light chain kinase is regulated by phosphorylation by PAK 1, a member of a family of protein kinases that is activated by the binding of the p21 GTPase. Although PAKs are generally thought to be involved in responding to stress, substrates for PAK's are not well characterized and myosin light chain kinase represents an important one. Based on extensive preliminary data, we propose that PAKs regulate the calcium sensitivity of smooth muscle contraction by a mechanism that involves the phosphorylation of myosin light chain kinase. We now propose experiments to test this hypothesis. The experiments described in Specific Aim 1 will investigate the kinetics of myosin light chain kinase phosphorylation by PAK 1, in vitro.
Specific Aim 2 will test the hypothesis by performing studies on skinned smooth muscles.
Specific Aim 3 will test the hypothesis by performing experiments on intact blood vessels. These experiments represent a powerful test of our hypothesis and they will provide important insights into the molecular mechanisms that regulate the calcium sensitivity of smooth muscle contraction.
| Gu, Lian-Zhi; Hu, Wen-Yang; Antic, Nenad et al. (2006) Inhibiting myosin light chain kinase retards the growth of mammary and prostate cancer cells. Eur J Cancer 42:948-57 |
| Fazal, Fabeha; Gu, Lianzhi; Ihnatovych, Ivanna et al. (2005) Inhibiting myosin light chain kinase induces apoptosis in vitro and in vivo. Mol Cell Biol 25:6259-66 |
| Nobe, Hiromi; Nobe, Koji; Fazal, Fabeha et al. (2003) Rho kinase mediates serum-induced contraction in fibroblast fibers independent of myosin LC20 phosphorylation. Am J Physiol Cell Physiol 284:C599-606 |
| Wirth, A; Schroeter, M; Kock-Hauser, C et al. (2003) Inhibition of contraction and myosin light chain phosphorylation in guinea-pig smooth muscle by p21-activated kinase 1. J Physiol 549:489-500 |
