Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities.
The well-known, muscle-specific smooth muscle myosin light chain kinase (MLCK) (smMLCK) and skeletal muscle MLCK (skMLCK) are dedicated protein kinases regulated by an autoregulatory segment C terminus of the catalytic core that blocks myosin regulatory light chain (RLC) binding and phosphorylation in the absence of Ca(2+)/calmodulin (CaM). Although it is known that a more recently discovered cardiac MLCK (cMLCK) is necessary for normal RLC phosphorylation in vivo and physiological cardiac performance, information on cMLCK biochemical properties are limited. We find that a fourth uncharacterized MLCK, MLCK4, is also expressed in cardiac muscle with high catalytic domain sequence similarity with other MLCKs but lacking an autoinhibitory segment. Its crystal structure shows the catalytic domain in its active conformation with a short C-terminal "pseudoregulatory helix" that cannot inhibit catalysis as a result of missing linker regions. MLCK4 has only Ca(2+)/CaM-independent activity with comparable Vmax and Km values for different RLCs. In contrast, the Vmax value of cMLCK is orders of magnitude lower than those of the other three MLCK family members, whereas its Km (RLC and ATP) and KCaM values are similar. In contrast to smMLCK and skMLCK, which lack activity in the absence of Ca(2+)/CaM, cMLCK has constitutive activity that is stimulated by Ca(2+)/CaM. Potential contributions of autoregulatory segment to cMLCK activity were analyzed with chimeras of skMLCK and cMLCK. The constitutive, low activity of cMLCK appears to be intrinsic to its catalytic core structure rather than an autoinhibitory segment. Thus, RLC phosphorylation in cardiac muscle may be regulated by two different protein kinases with distinct biochemical regulatory properties.
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390; audreyn.chang@utsouthwestern.edu.
Audrey N
0000-0002-0641-2685
AN
Chang
Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, United Kingdom;
Pravin
P
Mahajan
Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, United Kingdom; Institute for Pharmaceutical Chemistry, Goethe University Frankfurt, 60438 Frankfurt, Germany; Buchmann Institute for Life Sciences, Goethe University Frankfurt, 60438 Frankfurt, Germany;
Stefan
S
Knapp
Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;
Hannah
H
Barton
Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, FL 32610.
H Lee
HL
Sweeney
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390;
Kristine E
KE
Kamm
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390;
James T
JT
Stull
D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D051379:Mice; D008968:Molecular Conformation; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*
D018994:Myosin Light Chains; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase
MLCK4; cMLCK; calcium/calmodulin; crystallography; kinase