Mutation of serine 41 in the neuron-specific protein B-50 (GAP-43) prohibits phosphorylation by protein kinase C.
The neuron-specific, calmodulin-binding protein B-50 (also known as GAP-43, F1, or neuromodulin) is an endogenous substrate of protein kinase C (PKC). PKC exclusively phosphorylates Ser residues in B-50. As potential phosphorylation sites for PKC, Ser41, Ser110, and Ser122 were indicated, of which Ser41 is contained in the sequence ASF, which matches with the sequence of a synthetic PKC substrate. N-terminally 35S-labeled B-50, produced from cDNA, was subjected to digestion with Staphylococcus aureus V8 protease (SAP). Consecutively, 35S-labeled 28- and 15-kDa fragments were formed, similar to those after digestion of 32P-labeled B-50. In a previous study, we showed that the 32P-labeled 15-kDa SAP fragment contains all 32P radioactivity. The present data indicate that it contains the N-terminus of B-50 as well. The 15-kDa fragment, with a calculated length ranging from amino acid residue 1 to 65, contains only one potential PKC phosphorylation site, at Ser41. Mutagenesis of Ser41 into Thr or Ala resulted in recombinant B-50 products with mobilities on two-dimensional electrophoresis similar to those of the nonmutated recombinant B-50 and the rat brain B-50. Only [Ser41]B-50 was phosphorylated by PKC, whereas [Thr41]- or [Ala41]B-50 did not show any phosphorylation at the positions indicated on the immunoblots. This leads us to the conclusion that Ser41 is the sole phosphorylation site for PKC in vitro.
Nielander
H B
HB
Division of Molecular Neurobiology, Rudolf Magnus Institute, University of Utrecht, The Netherlands.
Schrama
L H
LH
van Rozen
A J
AJ
Kasperaitis
M
M
Oestreicher
A B
AB
Gispen
W H
WH
Schotman
P
P
D000818:Animals; D003001:Cloning, Molecular; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000235:genetics*; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D012694:Serine; D013379:Substrate Specificity
D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D012694:Serine; D011493:Protein Kinase C