Identification and cloning of Kidins220, a novel neuronal substrate of protein kinase D

Protein kinase D (PKD) is a serine/threonine kinase regulated by diacylglyc erol signaling pathways with unique domain composition and enzymatic proper ties, still awaiting identification of its specific substrate(s). Here we h ave isolated, cloned, and characterized a novel protein from PC12 cells, te rmed Kidins220 (kinase D-interacting substrate of 220 kDa), as the first id entified PKD physiological substrate. Kidins220 contains 11 ankyrin repeats and four transmembrane domains within the N-terminal region. We have shown that Kidins220 is an integral membrane protein selectively expressed in br ain and neuroendocrine cells, where it concentrates at the tip of neurites. In PC12 cells, PKD coimmunoprecipitates and phosphorylates endogenous Kidi ns220. This phosphorylation is increased after stimulating PKD activity in vivo by phorbol-12,13-dibutyrate treatment. A constitutively active PKD mut ant (PKD-S744E/S748E) phosphorylates recombinant Kidins220-VSVG in vitro in the absence of phorbol-12,13-dibutyrate. Conversely, Kidins220-VSVG phosph orylation is abolished when a dominant negative mutant of PHD (PKD-D733A) i s used. Moreover, a peptide within the Kidins220 sequence, containing serin e 919 in a consensus motif for PHD-specific phosphorylation, behaved as the best peptide substrate to date. Substitution of serine 919 to alanine abro gated peptide phosphorylation. Furthermore, by generating an antibody recog nizing Kidins220 phosphorylated on serine 919, we show that phorbol ester t reatment causes the specific phosphorylation of this residue in PC12 cells in vivo. Our results provide the first physiological substrate for PKD and indicate that Kidins220 is phosphorylated by PKD at serine 919 in vivo.