Submitted on April 11, 2006
Accepted on July 20, 2006
MYPT1 Mutants Demonstrate the Importance of aa 888-928
for the Interaction with PKGI
Allison M Given1, Ozgur Ogut1, and Frank V Brozovich1*
1 Cardiovascular Diseases, Mayo Clinic, Rochester, Minnesota, United States
* To whom correspondence should be addressed. E-mail: brozovich.frank{at}mayo.edu.
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 MLC20 and vasodilation. Others (23, 24) have suggested that the interaction of MYPT1 and PKGI is mediated by the COOH-terminal leucine zipper (LZ) of MYPT1 and the NH2-terminal LZ of PKGI, but we have previously shown that PKGI interacts with both LZ+ and LZ- MYPT1 isoforms (13). Interestingly, PKGI is known to preferentially bind to RR and RK motifs (6), and there is an RK motif within aa 888-928 of MYPT1 in both LZ+ and LZ-isoforms. Thus, to localize the domain of MYPT1 important for interaction of MYPT1 with PKGI, we designed four MYPT1 fragments that either contained both the aa 888-928 sequence and the downstream LZ domain, lacked both the aa 888-928 sequence and the LZ domain, lacked the aa 888-928 sequence, or lacked the LZ domain. Using co-immunoprecipitation, we found that only fragments containing the sequence at aa 888-928 (MYPT1FL and MYPT1TR2) were able to form a complex with PKGI in avian smooth muscle tissue lysates. Further, mutations of the RK motif at aa 916-917(R916K917) to AA decreased binding of MYPT1 to PKGI in chicken gizzard lysates while there was no effect of these mutations on binding in chicken aorta lysates. However, mutation of R916K917 to E916E917 eliminated binding, suggesting that one factor important for the interaction of PKGI with MYPT1 is the charge at aa 916-917. These results suggest that during cGMP-mediated signaling, aa 888-928 of MYPT1 mediate the interaction with PKGI.
