MYPT1 isoforms expressed in HEK293T cells are differentially phosphorylated after GTPγS treatment

Agonist stimulation of smooth muscle is known to activate RhoA/Rho kinase signaling, and Rho kinase phosphorylates the myosin targeting subunit (MYPT1) of myosin light chain (MLC) phosphatase at Thr696 and Thr853, which inhibits the activity of MLC phosphatase to produce a Ca2+ independent increase in MLC phosphorylation and force (Ca2+ sensitization). Alternative mRNA splicing produces four MYPT1 isoforms, which differ by the presence or absence of a central insert (CI) and leucine zipper (LZ). This study was designed to determine if Rho kinase differentially phosphorylates MYPT1 isoforms. In HEK293T cells expressing each of the four MYPT1 isoforms, we could not detect a change in Thr853 MYPT1 phosphorylation following GTPγS treatment. However, there is differential phosphorylation of MYPT1 isoforms at Thr696; GTPγS treatment increases MYPT1 phosphorylation for the CI+LZ- and CI-LZ- MYPT1 isoforms, but not the CI+LZ+ or CI-LZ+ MYPT1 isoforms. These data could suggest that in smooth muscle Rho kinase differentially phosphorylates MYPT1 isoforms.