Proteomic analysis of calcium/calmodulin-dependent protein kinase I and IV in vitro substrates reveals distinct catalytic preferences.
The multifunctional calcium/calmodulin-dependent protein kinases I and IV (CaMKI and CaMKIV) are closely related by primary sequence and predicted to have similar substrate specificities based on peptide studies. We identified a fragment of p300-(1-117) that is a substrate of both kinases, and through both mutagenesis and Edman phosphate ((32)P) release sequencing, established that CaMKI and CaMKIV phosphorylate completely different sites. The CaMKI site, Ser(89) ((84)LLRSGSSPNL(93)), fits the expected consensus whereas the CaMKIV site, Ser(24) ((19)SSPALSASAS(28)), is novel. To compare kinase substrate preferences more generally, we employed a proteomic display technique that allowed comparison of complex cell extracts phosphorylated by each kinase in a rapid in vitro assay, thereby demonstrating substrate preferences that overlapped but were clearly distinct. To validate this approach, one of the proteins labeled in this assay was identified by microsequencing as HSP25, purified as a recombinant protein, and examined as a substrate for both CaMKI and CaMKIV. Again, CaMKI and CaMKIV were different, this time in kinetics and stoichiometry of the phosphorylation sites, with CaMKI preferring Ser(15) ((10)LLRTPSWGPF(19)) to Ser(85) ((80)LNRQLSSGVS(89)) 3:1, but CaMKIV phosphorylating the two sites equally. These differences in substrate specificities emphasize the need to consider these protein kinases independently despite their close homology.
Corcoran
Ethan E
EE
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Joseph
James D
JD
MacDonald
Justin A
JA
Kane
Christopher D
CD
Haystead
Timothy A J
TA
Means
Anthony R
AR
D000595:Amino Acid Sequence; D000818:Animals; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002384:Catalysis; D050881:E1A-Associated p300 Protein; D006360:Heat-Shock Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D040901:Proteomics; D013379:Substrate Specificity; D015534:Trans-Activators / Q000378:metabolism
D006360:Heat-Shock Proteins; C486113:Hsbp1 protein, mouse; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D015534:Trans-Activators; D050881:E1A-Associated p300 Protein; C494361:Ep300 protein, mouse; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517598:Camk1 protein, mouse; C517685:Camk4 protein, mouse; C517681:Pnck protein, mouse