Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

Abstract

Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca(2+) sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V(max) and K(M) for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.

Scheme 1

Figure 1

Phosphoprotein and total protein detected in unphosphorylated (RLC, lane 1) and phosphorylated (RLCp, lane 2) MYL2. Images are made from the same 10% polyacrylamide gel successively treated with Pro-Q Diamond phosphoprotein and Sypro-Ruby total protein stains. Panel A: SYPRO Ruby stain for total protein detection. Panel B: Pro-Q Diamond stain for phosphoprotein detection.

Figure 2

Time course for smMLCK phosphorylation of RLC from various myosin isoforms. Unphosphorylated (RLC) and phosphorylated (RLCp) forms of the regulatory light chain are indicated. ELC, LC1, and LC2 are the essential light chains. Panel A: MYL2. Time points shown for lanes 1–6 are 0, 0.25, 0.5, 0.75, 1, and 10 min. Time points 1. and 10 minutes are separated on the gel by time points 2, 2.5, 3, 4, 5 min (not shown). Panel B: Porcine ventricular myosin. Time points shown for lanes 1–6 are 0, 10, 20, 40, 60, and 120 min. Panel C: Rabbit skeletal myosin. Time points identical to Panel B. Panel D: Chicken gizzard (smooth muscle) HMM. Time points shown for lanes 1–4 are 0, 5, 15, and 25 min. Running conditions are 10% polyacrylamide and 8M urea. Panels B–D show only the section of the gel with myosin ELC and RLC. The myosin heavy chain does not enter into the 10% polyacrylamide-urea gel. Phosphorylation rates are roughly similar for all samples tested except for skeletal myosin where smMLCK is unable to phosphorylate the RLC (see Table 1).

Figure 3

Time course for MYL2 phosphorylation by smMLCK at 3 MYL2 initial concentrations. Fitted curves were derived using Scheme 1. Initial concentrations for MYL2 given in the legend are after renormalization to account for detection dead-time. Original MYL2 concentrations are 12.5, 25, and 55 μM that correspond to 8, 16, and 36 μM, respectively, after renormalization. Kinetic constants are summarized in Table 1.