Binding of CaMKII to the giant muscle protein projectin: stimulation of CaMKII activity by projectin
| Autor: | Gernot Beinbrech, Michael Fährmann, Martin Erfmann |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Calmodulin
Immunoprecipitation Biophysics Muscle Proteins Grasshoppers In Vitro Techniques Biology Biochemistry Cytosol Myofibrils Ca2+/calmodulin-dependent protein kinase Myosin Animals Myocyte Phosphorylation Protein kinase A Molecular Biology Muscles musculoskeletal neural and ocular physiology musculoskeletal system Precipitin Tests Cell biology Enzyme Activation nervous system Calcium-Calmodulin-Dependent Protein Kinases cardiovascular system biology.protein Calcium-Calmodulin-Dependent Protein Kinase Type 2 Myofibril tissues |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - General Subjects. 1569:127-134 |
| ISSN: | 0304-4165 |
| DOI: | 10.1016/s0304-4165(01)00251-3 |
| Popis: | Projectin is an integral high molecular mass protein of insect flight muscle binding to myosin and paramyosin. Yet, the role of projectin in insect flight muscle is not well understood. In this study we provide evidence for the interaction of projectin with the calcium sensor Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Two CaMKII variants of 52 and 60 kDa, respectively, of locust flight muscle cells were shown by an anti-CaMKII antibody. Both variants were associated to myofibrils. The 52 kDa variant was also shown abundant in the cytosol. The cytosolic CaMKII variant was co-precipitated in vitro with externally added purified projectin in a dose-dependent manner. To specify the binding properties of CaMKII to projectin we used both purified projectin from the flight muscle of locust and CaMKII from rat forebrain, a naturally rich source of CaMKII. CaMKII is highly conserved even between insects and mammals. The binding of CaMKII to native projectin was demonstrated in vitro by the solid phase enzyme assay, immunoprecipitation, and 'overlay' binding. One mol projectin bound maximally 1.38 +/- 0.02 mol CaMKII in vitro with a K(d) of 3.08 Z+/- 0.09 nM. Application of in vitro autophosphorylated CaMKII revealed a decreased stoichiometry of binding to projectin (0.86 +/- 0.04 mol mol(-1)) accompanied by a lower affinity (K(d) of 5.54 +/- 0.73) compared to non-autophosphorylated CaMKII. Furthermore, the CaMKII phosphotransferase activity was stimulated up to 2-fold by projectin. Even in the presence of calmodulin projectin enhanced the CaMKII activity moderately. Our data suggest that projectin represents a subcellular compartment for CaMKII to achieve its specificity, and activity in insect flight muscle cells. |
| Databáze: | OpenAIRE |
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