Identification of calcium-regulated heat-stable protein of 24 kDa (CRHSP24) as a physiological substrate for PKB and RSK using KESTREL

A substrate for PKB alpha (protein kinase B alpha) was detected in liver extracts, and was purified and identified as CRHSP24 (calcium-regulated heat-stable protein of apparent molecular mass 24 kDa). PKB alpha, as well as SGK1 (serum- and glucocorticoid-induced protein kinase 1) and RSK (p90 ribosomal S6 kinase), phosphorylated CRHSP24 stoichiometrically at Ser(52) in vitro and its brain-specific isoform PIPPin at the equivalent residue (Ser(58)). CRHSP24 became phosphorylated at Ser(52) when HEK-293 (human embryonic kidney) cells were stimulated with IGF-1 (insulin-like growth factor-1) and this was prevented by inhibitors of PI3K (phosphoinositide 3-kinase), but not by rapamycin [an inhibitor of mTOR (mammalian target of rapamycin)] or PD 184352, an inhibitor of the classical MAPK (mitogen-activated protein kinase) cascade and hence the activation of RSK. IGF-1 induced a similar phosphorylation of CRHSP24 in ES (embryonic stem) cells from wild-type mice or mice that express the PDK1 (3-phosphoinositide-dependent kinase 1) mutant (PDK1 [L155E]) that activates PKB alpha normally, but cannot activate SGK. CRHSP24 also became phosphorylated at Ser(52) in response to EGF (epidermal growth factor) and this was prevented by blocking activation of both the classical MAPK cascade and the activation of PKB alpha, but not if just one of these pathways was inhibited. DYRK2 (dual-specificity tyrosine-phosphorylated and -regulated protein kinase 2) phosphorylated CRHSP24 at Ser(30), Ser(32) and Ser(41) in vitro, and Ser(41) was identified as a site phosphorylated in cells. These and other results demonstrate that CRHSP24 is phosphorylated at Ser(52) by PKB alpha in response to IGF-1, at Ser(52) by PKBa and RSK in response to EGF, and at Ser(41) in the absence of IGF-1/EGF by a DYRK isoform or another proline-directed protein kinase(s).