The key signal that contracts smooth muscle is an increase in [Ca2+]i which activates Ca2+/calmodulin-dependent myosin light chain kinase (MLCK), thereby promoting phosphorylation of myosin regulatory light chain (RLC) and initiating contraction. We investigate mechanisms necessary for activation of the smooth muscle contractile apparatus to unravel the complexities of interacting signaling networks. Is MLCK the only kinase that phosphorylates RLC in a Ca2+-dependent manner in bladder smooth muscle? Transgenic mice expressing biosensor MLCK are used to determine Ca2+-dependency of activation relative to [Ca2+]i , RLC phosphorylation and contraction. Protein transduction domain inhibitors are used to test involvement of specific kinases. Smooth muscle MLCK gene ablation will be restricted to smooth muscle cells by a tamoxifen-controlled ablation system for measurements of contractile responsiveness in isolated tissues and in vivo. Is MLCK targeting to the contractile domain of smooth muscle cells necessary and sufficient for RLC phosphorylation? Gene exons containing the actin-binding motifs and the myosin binding module will be deleted by knock-in procedures and contractile performance of smooth muscle will be characterized in vitro and in vivo. In skeletal muscle Ca2+ activation of contraction is mediated through a thin filament regulatory system, troponin-tropomyosin. Experiments in intact muscles show a correlation between RLC phosphorylation and post-tetanic potentiation of isometric force amplitude or treppe in fast-twitch skeletal muscles. The relative importance of RLC phosphorylation-force relationship was unclear because other factors may affect contractile performance in intact muscles including length-dependent effects on Ca2+ release from the sarcoplasmic reticulum, inter-myofilament spacing and calmodulin regulation of Ryr1, the calcium release channel. We therefore showed RLC phosphorylation and increased force amplitude are inhibited in fast-twitch skeletal muscle fibers from mice with ablation of the skeletal muscle MLCK gene. We have now developed procedures to isolate enzymatically dispersed mouse skeletal muscle fibers with biosensor molecules to investigate the temporal and spatial distributions of Ca2+/calmodulin activation of skeletal muscle MLCKs. The results from these investigations provide insights into Ca2+-dependent mechanisms recruited during exercise that enhance muscle performance. Phosphorylation of regulatory light chain (RLC) of sarcomeric myosin enhances ventricular contractions of the heart while phosphorylation of either sarcomeric or cytoplasmic myosin-IIB promotes sarcomere assembly. Increased Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) activity may lead to cardiomyopathy. Because cardiac myocytes contain both skeletal (sk) and smooth (sm) muscle MLCK, it is not clear if RLC phosphorylations are affected by one or both kinases. We propose to identify the kinase that phosphorylates the respective RLCs in addition to physiological roles for RLC phosphorylations in cardiac contraction and sarcomere assembly. We have overexpression and knockout models for both skMLCK and smMLCK specifically for cardiac myocytes. RLC phosphorylations are measured in cardiac tissues as well as isolated myocytes to determine functional consequences associated with changes in expression of specific MLCK isoforms. Anatomical properties of hearts from transgenic and knockout mice are also assessed. Sarcomere formation in response to hypertrophic agents and stresses are measured in myocytes from newborn transgenic or knockout mice.
RESEARCH INTERESTS
Response and Adaptation to Exercise
Myosin Light Chain Kinase Function in Smooth Muscle
Biochemical and biophysical mechanisms in signal integration and protein phosphorylation involving skeletal, heart and smooth muscle contractions
RECENT PUBLICATIONS
Kamm KE, Stull JT, "Dedicated myosin light chain kinases with diverse cellular functions" J Biol Chem, 276(7):4527-30, February 2001
Isotani E, Zhi G, Lau KS, Huang J, Mizuno Y, Persechini A, Geguchadze R, Kamm KE, Stull JT, "Real-time evaluation of myosin light chain kinase activation in smooth muscle tissues from a transgenic calmodulin-biosensor mouse." Proc Natl Acad Sci U S A, 101(16):6279-84, April 2004
Zhi G, Ryder JW, Huang J, Ding P, Chen Y, Zhao Y, Kamm KE, Stull JT, "Myosin light chain kinase and myosin phosphorylation effect frequency-dependent potentiation of skeletal muscle contraction." Proc Natl Acad Sci U S A, 102(48):17519-24, November 2005
Ryder, J. W., Lau, K. S., Kamm, K. E., and Stull, J. T., "Enhanced Skeletal Muscle Contraction with Myosin Light Chain Phosphorylation by a Calmodulin-sensing Kinase" J. Biol. Chem., 282:20447-20454, 2007
Jian Huang , John M. Shelton , James A. Richardson, Kristine E. Kamm, James T. Stull, "Myosin regulatory light chain phosphorylation attenuates cardiac hypertrophy" J. Biol. Chem, http://www.jbc.org/cgi/do May 2008
SIGNIFICANT PUBLICATIONS
Kamm KE, Stull JT, "Activation of smooth muscle contraction: relation between myosin phosphorylation and stiffness" Science, 232(4746):80-2, April 1986
Sweeney HL, Bowman BF, Stull JT, "Myosin light chain phosphorylation in vertebrate striated muscle: regulation and function" Am J Physiol, 264(5 Pt 1):C1085-95, May 1993
Smith L, Su X, Lin P, Zhi G, Stull JT, "Identification of a novel actin binding motif in smooth muscle myosin light chain kinase" J Biol Chem, 274(41):29433-8, October 1999
Kamm KE, Stull JT, "The function of myosin and myosin light chain kinase phosphorylation in smooth muscle" Annu Rev Pharmacol Toxicol, 25:593-620, 1985
Krueger JK, Olah GA, Rokop SE, Zhi G, Stull JT, Trewhella J, "Structures of calmodulin and a functional myosin light chain kinase in the activated complex: a neutron scattering study" Biochemistry, 36(20):6017-23, May 1997
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