Purification and characterization of a brain-specific protein kinase C substrate, neurogranin (p17). Identification of a consensus amino acid sequence between neurogranin and neuromodulin (GAP43) that corresponds to the protein kinase C phosphorylation site and the calmodulin-binding domain.
Neurogranin, formerly designated p17 (Baudier, J., Bronner, C., Kligman, D., and Cole, R. D.) (1989) J. Biol. Chem. 264, 1824-1828), a brain-specific in vitro substrate for protein kinase C (PKC), has been purified to homogeneity from bovine forebrain. The purified protein has a molecular mass of 7837.1 +/- 0.5 Da, determined by electrospray mass spectrometry. In the absence of reducing agent, dimers and higher oligomers accumulated. On sodium dodecyl sulfate-polyacrylamide gels the protein monomer migrated abnormally with an apparent molecular mass of 15,000-19,000 Da, depending on the percentage of polyacrylamide. The native protein is blocked at its amino terminus. The majority of the primary amino acid sequence was determined following proteolytic and chemical fragmentation. A comparison of the amino acid sequence of neurogranin with that of the brain-specific PKC substrate neuromodulin, revealed a strikingly conserved amino acid sequence AA(X)KIQA-SFRGH(X)(X)RKK(X)K. The two proteins are not related over the rest of their sequences. Neurogranin was shown to be phosphorylated in hippocampal slices incubated with 32Pi and phorbol esters stimulated neurogranin phosphorylation, suggesting that neurogranin is likely to be an in vivo substrate for PKC. In vitro phosphorylation of neurogranin by PKC produced a shift of the isoelectric point of the protein (pI 5.6) to a more acidic value (pI 5.4). Tryptic digestion of the phosphorylated protein yielded a single phosphopeptide having the sequence IQASFR, where the serine residue is the phosphorylated amino acid. This phosphopeptide is part of the conserved sequence shared with neuromodulin and also corresponds to the PKC phosphorylation site on neuromodulin (Apel, E. D., Byford, M. F., Au, D., Walsh, K. A., and Storm, D. R. (1990) Biochemistry 29, 2330-2335). Evidence was obtained suggesting that neurogranin binds to calmodulin in the absence of Ca2+, a feature that also characterizes neuromodulin. We propose that the amino acid sequence shared by neurogranin and neuromodulin reflects a functional relationship between these two proteins and that the consensus sequence represents a conserved PKC phosphorylation site and a calmodulin binding domain that characterizes a class of brain-specific PKC substrates.
Baudier
J
J
Centre de Neuorchimie du Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale Unité 44, Strasbourg, France.
Deloulme
J C
JC
Van Dorsselaer
A
A
Black
D
D
Matthes
H W
HW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D019922:GAP-43 Protein; D007700:Kinetics; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051579:Neurogranin; D010748:Phosphopeptides / Q000302:isolation & purification; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; D051579:Neurogranin; D011493:Protein Kinase C