During nitric oxide signaling, type Ialpha cGMP-dependent protein kinase (PKGIalpha) 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-PKGIalpha interaction is mediated by the COOH-terminal leucine zipper (LZ) of MYPT1 and the NH(2)-terminal LZ of PKGIalpha (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 PKGIalpha interacts with LZ-positive (LZ+) and LZ-negative (LZ-) MYPT1 isoforms (13). Interestingly, PKGIalpha 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-PKGIalpha 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 PKGIalpha in avian smooth muscle tissue lysates. Furthermore, mutations of the RK motif at aa 916-917 (R(916)K(917)) to AA decreased binding of MYPT1 to PKGIalpha in chicken gizzard lysates; these mutations had no effect on binding in chicken aorta lysates. However, mutation of R(916)K(917) to E(916)E(917) eliminated binding, suggesting that one factor important for the PKGIalpha-MYPT1 interaction is the charge at aa 916-917. These results suggest that, during cGMP-mediated signaling, aa 888-928 of MYPT1 mediate the PKGIalpha-MYPT1 interaction.