Molecular cloning of bovine cardiac muscle heat-shock protein 70 kDa and its phosphorylation by cAMP-dependent protein kinase in vitro.
The 70-kDa heat-shock protein (Hsp70) has been cloned and sequenced from bovine cardiac muscle. On the basis of sequence features, the gene corresponds to the cytoplasmic form of Hsp70. This cardiac Hsp70 cDNA clone has an open reading frame of 1926 bp coding for 641 amino acids and a predicted molecular mass of 70.25 kDa. Comparison of the amino acid sequence revealed an extensive sequence identity with other species of Hsp70. Escherichia coli expressed cardiac Hsp70 stimulated a 2-fold increase in calcineurin (CaN) activity. Notably, we observed that Hsp70 directly interacts with CaN using a pull-down assay. Furthermore, expressed cardiac-specific Hsp70 was phosphorylated in vitro by cAMP-dependent protein kinase. Phosphorylation resulted in the incorporation of 0.1 mol of phosphate per mol of Hsp70. The phosphorylated Hsp70 was unable to activate the phosphatase activity of CaN. This is the first demonstration that Hsp70 is phosphorylated by cAMP-dependent protein kinase and provides an on/off switch for the regulation of CaN signaling by Hsp70.
Lakshmikuttyamma
Ashakumary
A
Department of Pathology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 4H4, Canada.
Selvakumar
Ponniah
P
Anderson
Deborah H
DH
Datla
Raju S
RS
Sharma
Rajendra K
RK
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019703:Calcineurin / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D018840:HSP70 Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D019703:Calcineurin