Phosphorylation screening identifies translational initiation factor 4GII as an intracellular target of Ca(2+)/calmodulin-dependent protein kinase I.
CaMKI is a Ca2+/calmodulin-dependent protein kinase that is widely expressed in eukaryotic cells and tissues but for which few, if any, physiological substrates are known. We screened a human lung cDNA expression library for potential CaMKI substrates by solid phase in situ phosphorylation ("phosphorylation screening"). Multiple overlapping partial length cDNAs encoding three proteins were detected. Two of these proteins are known: 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase and eukaryotic translation initiation factor (eIF) 4GII. To determine whether CaMKI substrates identified by phosphorylation screening represent authentic physiological targets, we examined the potential for [Ca2+]i- and CaMKI-dependent phosphorylation of eIF4GII in vitro and in vivo. Endogenous eIF4GII immunoprecipitated from HEK293T cells was phosphorylated by CaMKI, in vitro as was a recombinant fragment of eIF4GII encompassing the central and C-terminal regions. The latter phosphorylation occurred with favorable kinetics (Km = 1 microm; kcat = 1.8 s-1) at a single site, Ser1156, located in a segment of eIF4GII aligning with the phosphoregion of eIF4GI. Phosphopeptide mapping and back phosphorylation experiments revealed [Ca2+]i-dependent, CaMKI site-specific, eIF4GII phosphorylation in vivo. This phosphorylation was blocked by kinase-negative CaMKI consistent with a requirement for endogenous CaMKI for in vivo eIF4GII phosphorylation. We conclude that phosphorylation screening is an effective method for searching for intracellular targets of CaMKI and may have identified a new role of Ca2+ signaling to the translation apparatus.
D000595:Amino Acid Sequence; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D017931:DNA Primers; D039642:Eukaryotic Initiation Factors / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D039642:Eukaryotic Initiation Factors; D012333:RNA, Messenger; D017871:Calcium-Calmodulin-Dependent Protein Kinases