MYPT1 mutants demonstrate the importance of aa 888-928 for the interaction with PKGIα

Allison M. Given, Ozgur Ogut, Frank V. Brozovich

Research output: Contribution to journalArticle

31 Scopus citations

Abstract

During nitric oxide signaling, type Iα cGMP-dependent protein kinase (PKGIα) 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-PKGIα interaction is mediated by the COOH-terminal leucine zipper (LZ) of MYPT1 and the NH2-terminal LZ of PKGIα (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 PKGIα interacts with LZ-positive (LZ+) and LZ-negative (LZ-) MYPT1 isoforms (13). Interestingly, PKGIα 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-PKGIα 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 PKGIα in avian smooth muscle tissue lysates. Furthermore, mutations of the RK motif at aa 916-917 (R916K 917) to AA decreased binding of MYPT1 to PKGIα in chicken gizzard lysates; these mutations had no effect on binding in chicken aorta lysates. However, mutation of R916K917 to E 916E917 eliminated binding, suggesting that one factor important for the PKGIα-MYPT1 interaction is the charge at aa 916-917. These results suggest that, during cGMP-mediated signaling, aa 888-928 of MYPT1 mediate the PKGIα-MYPT1 interaction.

Original languageEnglish (US)
Pages (from-to)C432-C439
JournalAmerican Journal of Physiology - Cell Physiology
Volume292
Issue number1
DOIs
StatePublished - Jan 1 2007

Keywords

  • Calcium desensitization
  • Myosin light chain phosphatase
  • Nitric oxide
  • Smooth muscle
  • cGMP
  • cGMP-dependent protein kinase

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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