Real-time in vivo proteomic identification of novel kinase substrates in smooth muscle.
Relaxation of smooth muscle can occur through agonists (such as nitric oxide) that activate guanylyl cyclase and stimulate the production of cGMP, activating its target, cGMP-dependent protein kinase (PKG). This kinase can raise the Ca2+ threshold for contraction, thus causing Ca2+ desensitization, but the mechanism for this event is not completely understood. Ca2+ sensitization/desensitization pathways are essential for maintenance of normal smooth muscle tone, and abnormalities in these pathways have been shown to be key components in the pathogenesis of diseases such as hypertension and asthma in humans. Our laboratory has devised a proteomic method to specifically address the question of what proteins are early phosphorylation targets in calcium desensitization. Using ileum smooth muscle, we metabolically labeled the muscle with (32P)-orthophosphate, permeabilized the muscle, established constant calcium concentrations, and stimulated with 8-bromo-cGMP, which activates PKG. Proteins whose phosphorylation state changed in response to cGMP at constant levels of calcium were separated with two-dimensional gel electrophoresis, identified by autoradiography, and sequenced with nanospray mass spectrometry. Using this technique, we identified a previously uncharacterized PKG phosphoprotein, which we have termed CHASM (Calponin Homology Smooth Muscle protein). Using physiological muscle bath contraction studies, we have validated CHASM as a component of calcium desensitization pathways in smooth muscle.
Wooldridge
Anne A
AA
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA.
Haystead
Timothy A
TA
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011506:Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011817:Rabbits; D016415:Sequence Alignment
D010750:Phosphoproteins; D011506:Proteins; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP; D002118:Calcium