Proteomic Analysis of Calcium/Calmodulin-dependent Protein Kinase I and IV in Vitro Substrates Reveals Distinct Catalytic Preferences*210

Abstract

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 (³²P) release sequencing, established that CaMKI and CaMKIV phosphorylate completely different sites. The CaMKI site, Ser⁸⁹(⁸⁴LLRSGSSPNL⁹³), fits the expected consensus whereas the CaMKIV site, Ser²⁴(¹⁹SSPALSASAS²⁸), 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 vitroassay, 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 Ser¹⁵(¹⁰LLRTPSWGPF¹⁹) to Ser⁸⁵ (⁸⁰LNRQLSSGVS⁸⁹) 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.