Phosphoprotein B-50: localization of proteolytic sites for S. aureus V8 protease using truncated cRNAs for cell-free translation.
B-50 (= GAP-43, F1, and P-57 or neuromodulin) is a nervous tissue-specific, growth-associated protein, localized in the presynaptic membrane. Phosphorylation by protein kinase C at Ser41 appears to play a role in B-50/calmodulin interaction and neurotransmitter release. Previous studies have shown that digestion of the phosphorylated protein with S. aureus V8 protease (SAP) resulted consecutively in 28- and 15-kDa phospho fragments, the latter containing all incorporated phosphate. These proteolytic products of digestion with SAP have frequently been used to identify B-50 in various systems. Therefore we were interested to find out the location of these fragments in the rat B-50 molecule. For this purpose, the rat cDNA for B-50 was used to generate full-length and truncated cRNAs for cell-free translation. B-50 and B-50 peptides were either N-terminally labeled with [35S]methionine (residues 1 and 5) as a tracer, or they were phosphorylated in vitro by protein kinase C. SAP digestion of the immunoprecipitated, 35S-labeled translation products produced similar 28- and 15-kDa fragments as were obtained from 32P-labeled B-50, indicating that these fragments are N-terminal. Relative mobilities of the N-terminal B-50 fragments of known length were used as internal standards for the calculation of the length of SAP and phospho fragments. Comparing the 35S- and 32P-labeled products, four SAP sites at Glu12, Glu28, Glu65, and Glu132 could be deduced. The latter two sites are in accordance with sequence data of C-terminal fragments from the literature. All available data could be fitted into one scheme.
Nielander
H B
HB
Division of Molecular Neurobiology, Rudolf Magnus Institute, Utrecht, The Netherlands.
Van Rozen
A J
AJ
Schrama
L H
LH
Kasparaitis
M
M
Oestreicher
A B
AB
Gispen
W H
WH
Schotman
P
P
D000595:Amino Acid Sequence; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002474:Cell-Free System; D004247:DNA / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000378:metabolism; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D018075:RNA, Complementary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*
D002148:Calmodulin-Binding Proteins; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D018075:RNA, Complementary; D011993:Recombinant Fusion Proteins; D012313:RNA; D004247:DNA; D004262:DNA Restriction Enzymes; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase