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 (PKC) substrate enriched in nerve growth cones that has been implicated in growth cone plasticity. Here we investigated whether B-50 is a physiological substrate for casein kinase II (CKII) in purified rat cortical growth cone preparations. Using site-specific proteolysis and known modulators of PKC, in combination with immunoprecipitation, mass spectrometry, and phosphoamino acid analysis, we demonstrate that endogenous 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 proteolytic fragments containing Ser41. Under basal conditions, however, phosphorylation was predominantly associated with fragments not containing Ser41. 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 immunoprecipitated chick B-50 generated a 4-kDa C-terminal B-50 phosphopeptide, confirming that phosphorylation of the CKII domain occurs across evolutionary diverse species. We conclude that B-50 in growth cones is not only a substrate for PKC, but also for CKII.
Edgar
M A
MA
Rudolf Magnus Institute for Neurosciences, Department of Medical Pharmacology, University of Utrecht, The Netherlands.
Pasinelli
P
P
DeWit
M
M
Anton
B
B
Dokas
L A
LA
Pastorino
L
L
DiLuca
M
M
Cattabeni
F
F
Gispen
W H
WH
De Graan
P N
PN
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001369:Axons / Q000378:metabolism*; D047390:Casein Kinase II; D002417:Cattle; D002540:Cerebral Cortex / Q000378:metabolism*; D002645:Chickens; D019922:GAP-43 Protein / Q000737:chemistry* / Q000378:metabolism*; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D016548:Prosencephalon / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D016415:Sequence Alignment; D013379:Substrate Specificity
D019922:GAP-43 Protein; D010446:Peptide Fragments; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases