HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 47, 32860-32869, November 21, 2008

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 283/47/32860    most recent M804916200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sharma, A. K. Articles by Rigby, A. C. Search for Related Content PubMed PubMed Citation Articles by Sharma, A. K. Articles by Rigby, A. C. Social Bookmarking                      What's this?

Probing the Interaction between the Coiled Coil Leucine Zipper of cGMP-dependent Protein Kinase I and the C Terminus of the Myosin Binding Subunit of the Myosin Light Chain Phosphatase^*

Alok K. Sharma, Guo-Ping Zhou, Joseph Kupferman, Howard K. Surks, Eva N. Christensen, James J. Chou^¶, Michael E. Mendelsohn, and Alan C. Rigby¹

From the Divison of Molecular and Vascular Medicine, Center for Vascular Biology Research, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, Molecular Cardiology Research Institute, Tufts Medical Center, Boston, Massachusetts 02111, and ^¶Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

Nitric oxide and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase I (PKG-I)-mediated activation of myosin phosphatase (MLCP). Mechanistically it has been proposed that protein-protein interactions between the N-terminal leucine zipper (LZ) domain of PKG-I ((PKG-I_1-59) and the LZ and/or coiled coil (CC) domain of the myosin binding subunit (MBS) of MLCP are localized in the C terminus of MBS. Although recent studies have supported these interactions, the critical amino acids responsible for these interactions have not been identified. Here we present structural and biophysical data identifying that the LZ domain of PKG-I_1-59 interacts with a well defined 42-residue CC motif (MBS_CT42) within the C terminus of MBS. Using glutathione S-transferase pulldown experiments, chemical cross-linking, size exclusion chromatography, circular dichroism, and isothermal titration calorimetry we identified a weak dimer-dimer interaction between PKG-I_1-59 and this C-terminal CC domain of MBS. The K_d of this non-covalent complex is 178.0 ± 1.5 µM. Furthermore our ¹H-¹⁵N heteronuclear single quantum correlation NMR data illustrate that this interaction is mediated by several PKG-I residues that are on the a, d, e, and g hydrophobic and electrostatic interface of the C-terminal heptad layers 2, 4, and 5 of PKG-I. Taken together these data support a role for the LZ domain of PKG-I and the CC domain of MBS in this requisite contractile complex.

Received for publication, June 27, 2008 , and in revised form, August 25, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants 1PO1HL077378 (to M. E. M.) and 1PO1HL077378 and 1RO1HL74069 (to H. K. S.). This work was also supported by an Atorvastatin Research Award (to A. C. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. SI1-SI4.

¹ To whom correspondence should be addressed: Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA, 02215. Tel.: 617-667-0637; Fax: 617-975-5505; E-mail: arigby{at}bidmc.harvard.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?