PHOSPHORYLATION OF THE CASEIN KINASE-II DOMAIN OF B-50 (GAP-43) IN RAT CORTICAL GROWTH CONES

Growth-associated phosphoprotein B-50 is a neural protein kinase C (PK C) substrate enriched in nerve growth cones that has been implicated i n growth cone plasticity. Here we investigated whether B-50 is a physi ological substrate for casein kinase II (CKII) in purified rat cortica l growth cone preparations. Using site-specific proteolysis and known modulators of PKC, in combination with immunoprecipitation, mass spect rometry, and phosphoamino acid analysis, we demonstrate that endogenou s growth cone B-50 is phosphorylated at multiple sites, on both serine and threonine residues. Consistent with previous reports, stimulation of PKC activity increased the phosphorylation of only those proteolyt ic fragments containing Ser(41). Under basal conditions, however, phos phorylation was predominantly associated with fragments not containing Ser(41). Mass spectrometry of tryptic digests of B-50, which had been immunoprecipitated from untreated growth cones, revealed that in situ phosphorylation occurs within peptides B-50(181-198) and B-50(82-98). These peptides contain the major and minor in vitro CKII phosphosites , respectively. In addition, cyanogen bromide digestion of immunopreci pitated chick 8-50 generated a 4-kDa C-terminal B-50 phosphopeptide, c onfirming that phosphorylation of the CKII domain occurs across evolut ionary diverse species. We conclude that B-50 in growth cones is not o nly a substrate for PKC, but also for CKII.