Dimerization of cGMP-dependent protein kinase 1alpha and the myosin-binding subunit of myosin phosphatase: role of leucine zipper domains.
Nitric oxide (NO) and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase (cGK)-mediated activation of myosin phosphatase, which dephosphorylates myosin light chains. We recently found that cGMP-dependent protein kinase 1alpha binds directly to the myosin-binding subunit (MBS) of myosin phosphatase via the leucine/isoleucine zipper of cGK. We have now studied the role of the leucine zipper domain of MBS in dimerization with cGK and the leucine/isoleucine zipper and leucine zipper domains of both proteins in homodimerization. Mutagenesis of the MBS leucine zipper domain disrupts cGKIalpha-MBS dimerization. Mutagenesis of the MBS leucine zipper eliminates MBS homodimerization, while similar disruption of the cGKIalpha leucine/isoleucine zipper does not prevent formation of cGK dimers. The MBS leucine zipper domain is phosphorylated by cGK, but this does not have any apparent effect on heterodimer formation between the two proteins. MBS LZ mutants that are unable to bind cGK were poor substrates for cGK. These data support the theory that the MBS leucine zipper domain is necessary and sufficient to mediate both MBS homodimerization and binding of the protein to cGK. In contrast, the leucine/isoleucine zipper of cGK is required for binding to MBS, but not for cGK homodimerization. These data support that the MBS and cGK leucine zipper domains mediate the interaction between these two proteins. The contribution of these domains to both homodimerization and their specific interaction with each other suggest that additional regulatory mechanisms involving these domains may exist.
Surks
Howard K
HK
Molecular Cardiology Research Institute, Cardiology Division, Department of Medicine, New England Medical Center Hospitals, Inc., Tufts University School of Medicine, 750 Washington Street, Box 80, Boston, MA 02111, USA. HSurks@tufts-memc.org
Mendelsohn
Michael E
ME
D000595:Amino Acid Sequence; D000818:Animals; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D016350:Leucine Zippers / Q000502:physiology; D008969:Molecular Sequence Data; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism*; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; D043263:Myosin-Light-Chain Phosphatase; D009218:Myosins