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MUSCLE CELL BIOLOGY AND CELL MOTILITY

MYPT1 mutants demonstrate the importance of aa 888–928 for the interaction with PKGI

Division of Cardiovascular Diseases, Mayo Clinic, Rochester, Minnesota

Submitted 11 April 2006 ; accepted in final form 20 July 2006

During nitric oxide signaling, type I cGMP-dependent protein kinase (PKGI) activates myosin light chain (MLC) phosphatase through an interaction with the 130-kDa myosin targeting subunit (MYPT1), leading to dephosphorylation of 20-kDa MLC and vasodilatation. It has been suggested that the MYPT1-PKGI interaction is mediated by the COOH-terminal leucine zipper (LZ) of MYPT1 and the NH₂-terminal LZ of PKGI (HK Surks and ME Mendelsohn. Cell Signal 15: 937–944, 2003; HK Surks et al. Science 286: 1583–1587, 1999), but we previously showed that PKGI interacts with LZ-positive (LZ+) and LZ-negative (LZ–) MYPT1 isoforms (13). Interestingly, PKGI is known to preferentially bind to RR and RK motifs (WR Dostmann et al. Proc Natl Acad Sci USA 97: 14772–14777, 2000), and there is an RK motif within the aa 888–928 sequence of MYPT1 in LZ+ and LZ– isoforms. Thus, to localize the domain of MYPT1 important for the MYPT1-PKGI interaction, we designed four MYPT1 fragments that contained both the aa 888–928 sequence and the downstream LZ domain (MYPT1FL), lacked both the aa 888–928 sequence and the LZ domain (MYPT1TR), lacked only the aa 888–928 sequence (MYPT1SO), or lacked only the LZ domain (MYPT1TR2). Using coimmunoprecipitation, we found that only the fragments containing the aa 888–928 sequence (MYPT1FL and MYPT1TR2) were able to form a complex with PKGI in avian smooth muscle tissue lysates. Furthermore, mutations of the RK motif at aa 916–917 (R⁹¹⁶K⁹¹⁷) to AA decreased binding of MYPT1 to PKGI in chicken gizzard lysates; these mutations had no effect on binding in chicken aorta lysates. However, mutation of R⁹¹⁶K⁹¹⁷ to E⁹¹⁶E⁹¹⁷ eliminated binding, suggesting that one factor important for the PKGI-MYPT1 interaction is the charge at aa 916–917. These results suggest that, during cGMP-mediated signaling, aa 888–928 of MYPT1 mediate the PKGI-MYPT1 interaction.

myosin light chain phosphatase; nitric oxide; smooth muscle; calcium desensitization; cGMP-dependent protein kinase; cGMP