Amino acid sequences surrounding the cAMP-dependent and calcium/calmodulin-dependent phosphorylation sites in rat and bovine synapsin I.

Abstract

The amino acid sequences surrounding three major phosphorylation sites in rat and bovine synapsin I have been determined by employing automated gas-phase sequencing and manual Edman degradation of purified phosphopeptide fragments. Site 1 is a serine residue phosphorylated by cAMP-dependent protein kinase and by calcium/cahuodulindependent protein kinase I. The sequence around site 1 was derived from tryptic/chymotryptic phosphopeptides and overlapping cyanogen bromide cleavage fragments. This sequence, identical in rat and bovine synapsin I, is Asn-Tyr-Leu-ArgArg-Arg-Leu-Ser(P)-Asp-Ser-Asn-Phe-Met. Site 1 is located at the NH2 terminus of the protein, within the collagenaseresistant head region. Sites 2 and 3 are serine residues phosphorylated by calcium/calmodulin-dependent protein kinase II. The sequences surrounding bovine site 2 and site 3 were derived from tryptic phosphopeptides and overlapping fragments generated by cleavage with chymotrypsin, collagenase, and endoproteinase Lys-C. The sequence around bovine site 2 is Thr-Arg-Gln-Thr-Ser(P)-Val-Ser-Gly-Gln-Ala-ProPro-Lys, and the sequence around bovine site 3 is Thr-ArgGln-Ala-Ser(P)-Gln-Ala-Gly-Pro-Met-Pro-Arg. Sites 2 and 3 are located within the COOH-terminal, collagenase-sensitive tail region of the molecule, separated by 36 amino acids. The sequences surrounding rat site 2 and site 3 were derived from tryptic phosphopeptides. The sequence around rat site 2 is Gln-Ala-Ser(P)-Ile-Ser-Gly-Pro-Ala-Pro-Pro-Lys, and the sequence around rat site 3 is Gln-Ala-Ser(P)-Gln-Ala-Gly-ProGly-Pro-Arg. Thus, the sequences surrounding the four sites that are phosphorylated by calcium/calmodulin-dependent protein kinase II, namely sites 2 and 3 in rat and bovine synapsin I, exhibit a high degree of homology.

Similar substrate recognition motifs for mammalian AMP‐activated protein kinase, higher plant HMG‐CoA reductase kinase‐A, yeast SNF1, and mammalian calmodulin‐dependent protein kinase I

“…For CaMKI, the recognit~gn motif defined in this and the previous study [18] is consiswnt with the two protein substrates for which sequence inforn~ation is available, i.e. synapsin I (LRRRLSDSNF [29]) and t!le cAMP-response element binding protein, CREB (LSRRPS__YRKI [30]). Indeed, L at P-5, R at P-3 and I or F at I ~ + 4 is the optimal combination of residues for CaMKI as j~tdged by the results in Table 2.…”

Calcium/Calmodulin-Dependent Protein Kinase II

“…In contrast, cAMP-kinase [52] and Ca2+/calmodulin-dependent protein kinase I [54] phosphorylate synapsin I at a distinct site (site 1). Analysis of the phosphorylation sites in bovine and rat synapsin [53] reveal that sites 2 and 3 conform to the consensus phosphorylation sequence for CaM-kinase II. Synapsin I is thought to play a critical role in the release of neurotransmitter at nerve synaptic vesicles and inhibits neurotransmitter exocytosis by interfering with vesicle movement or fusion with the plasma membrane.…”

“…Phosphorylation can attain a stoichiometry of approx. 1.8 mol/mol in two sites (2 and 3) in the collagenasesensitive domain of synapsin I [52,53]. In contrast, cAMP-kinase [52] and Ca2+/calmodulin-dependent protein kinase I [54] phosphorylate synapsin I at a distinct site (site 1).…”

Detection by chemical cross-linking of bovine brain synapsin I self-association

“…Such a similarity has been pointed out by Czernik et al (1987) for the peptides corresponding to the different phosphorylation sites. In addition, Petrucci et al (1988) have reported that human and bovine brain synapsin I appear to share at least 88 0 sequence identity.…”

“…Synapsin I consists of two domains: a N-terminal collagenase-resistant domain and a proline-rich C-terminal domain susceptible to degradation by collagenase. Its primary structure has been deduced from rat brain cDNA (McCaffery & De Gennaro, 1986) and recently partially modified (Czernik et al, 1987). In addition, synapsin I can be phosphorylated either by cyclic-AMPdependent protein kinase and Ca2+/calmodulin-dependent protein kinase I at site 1 in the N-terminal region of the molecule or by Ca2+/calmodulin-dependent protein kinase II at sites 2 and 3 in the collagenase-sensitive region of the molecule.…”