Interactions between the leucine-zipper motif of cGMP-dependent protein kinase and the C-terminal region of the targeting subunit of myosin light chain phosphatase.
Nitric oxide induces vasodilation by elevating the production of cGMP, an activator of cGMP-dependent protein kinase (PKG). PKG subsequently causes smooth muscle relaxation in part via activation of myosin light chain phosphatase (MLCP). To date, the interaction between PKG and the targeting subunit of MLCP (MYPT1) is not fully understood. Earlier studies by one group of workers showed that the binding of PKG to MYPT1 is mediated by the leucine-zipper motifs at the N and C termini, respectively, of the two proteins. Another group, however, reported that binding of PKG to MYPT1 did not require the leucine-zipper motif of MYPT1. In this work we fully characterized the interaction between PKG and MYPT1 using biophysical techniques. For this purpose we constructed a recombinant PKG peptide corresponding to a predicted coiled coil region that contains the leucine-zipper motif. We further constructed various C-terminal MYPT1 peptides bearing various combinations of a predicted coiled coil region, extensions preceding this coiled coil region, and the leucine-zipper motif. Our results show, firstly, that while the leucine-zipper motif at the N terminus of PKG forms a homodimeric coiled coil, the one at the C terminus of MYPT1 is monomeric and non-helical. Secondly, the leucine-zipper motif of PKG binds to that of MYPT1 to form a heterodimer. Thirdly, when the leucine-zipper motif of MYPT1 is absent, the PKG leucine-zipper motif binds to the coiled coil region and upstream segments of MYPT1 via formation of a heterotetramer. These results provide rationalization of some of the findings by others using alternative binding analyses.
Lee
Eunhee
E
Cardiovascular Program, Boston Biomedical Research Institute, Watertown, MA 02472, USA.
Hayes
David B
DB
Langsetmo
Knut
K
Sundberg
Eric J
EJ
Tao
Terence C
TC
D000818:Animals; D001665:Binding Sites; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D016350:Leucine Zippers; D043263:Myosin-Light-Chain Phosphatase / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits
D021122:Protein Subunits; D017869:Cyclic GMP-Dependent Protein Kinases; D043263:Myosin-Light-Chain Phosphatase