Cardiac myosin light chain is phosphorylated by Ca ²⁺ /calmodulin-dependent and -independent kinase activities

Abstract

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.

Role of myosin light chain phosphatase in cardiac physiology and pathophysiology

“…In normal beating hearts, Serine 15 is 40% phosphorylated (0.40 mol phosphate/mol RLC) which is greater than the 10% or lower phosphorylation normally observed in resting skeletal and smooth muscles (Fig. 1) [13, 15-17, 21-23]. …”

“…The MLCK family is part of a large group of Ca 2+ /calmodulin (CaM)-dependent protein kinases [60], and is comprised of four distinct kinases, MLCK1, 2, 3, and 4, which are each encoded by distinct genes ( MYLK1 , MYLK2 , MYLK3 , and MYLK4 ) [13, 17, 23]. Based on their muscle-type specific expression and activities, MLCK1 is known as smooth muscle MLCK (smMLCK), MLCK2 as skeletal muscle MLCK (skMLCK), and MLCK3 as cardiac muscle MLCK (cMLCK).…”

“…Based on their muscle-type specific expression and activities, MLCK1 is known as smooth muscle MLCK (smMLCK), MLCK2 as skeletal muscle MLCK (skMLCK), and MLCK3 as cardiac muscle MLCK (cMLCK). MLCK4 has only recently been characterized, and is potentially another cardiac muscle MLCK based on its selective protein expression in cardiac myocytes [23]. …”

Angiotensin II Facilitates Matrix Metalloproteinase-9-Mediated Myosin Light Chain Kinase Degradation in Pressure Overload-Induced Cardiac Hypertrophy

“…Although Ang II did not play a direct role in regulating MLCP, cultured cardiomyocytes with adenovirus-mediated overexpression of MYPT2 and PP1cδ could rapidly block the Ang II-induced sarcomere organization [16]. It has been reported that MLC2 could be phosphorylated by MLCK in Ca 2+ /CaM-dependent and Ca 2+ /CaM-independent manners [20]. Our study revealed that Ang II promoted CaM expression, which may partially account for the increase of phosphorylated MLC2 levels after acute Ang II administration.…”

“…However, recent studies suggested that the catalytic activity of MLCK does not appear to be regulated by Ca 2+ /calmodulin [19, 20]. Several matrix metalloproteinases (MMPs), especially matrix metalloproteinase 9 (MMP9), have been reported to play an important role during the progression of cardiac disease by regulate the expression of proteins and degradation of kinases [21-23].…”