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* This work was supported in part by Grants-in-Aid from the Fujita Health University, Grants-in-Aid for Scientific Research (C) (06680773), and Grants-in-Aid for Scientific Research on Priority Areas (06253218, 06276218, 07268221, and 07279242) from the Ministry of Education, Science and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § A Postdoctoral Fellow of the Japan Society for the Promotion of Science. ¶ Present address: Nippi Research Institute of Biomatrix, Adachi-ku, Tokyo 120, Japan.
Posttranslational modifications of synapsin I, a major phosphoprotein in synaptic terminals, were studied by mass spectrometry. In addition to a well known phosphorylation site by calmodulin-dependent protein kinase II (CaM kinase II), a hitherto unrecognized site (Ser553) was found phosphorylated in vivo. The phosphorylation site is immediately followed by a proline, suggesting that the protein is an in vivo substrate of so-called proline-directed protein kinase(s). To identify the kinase involved, three proline-directed protein kinases expressed highly in the brain, i.e. mitogen-activated protein (MAP) kinase, Cdk5-p23, and glycogen synthase kinase 3β, were tested for the in vitro phosphorylation of synapsin I. Only MAP kinase and Cdk5-p23 phosphorylated synapsin I stoichiometrically. The phosphorylation sites were determined to be Ser551 and Ser553 with Cdk5-p23, and Ser62, Ser67, and Ser551 with MAP kinase. Upon phosphorylation with MAP kinase, synapsin I showed reduced F-actin bundling activity, while no significant effect on the interaction was observed with the protein phosphorylated with Cdk5-p23. These results raise the possibility that the so-called proline-directed protein kinases together with CaM kinase II and cAMP-dependent protein kinase play an important role in the regulation of synapsin I function.
Synapsin I has been characterized as one of the major phosphoproteins in nerve terminals and is thought to be involved in the regulation of neurotransmitter release (for reviews see Refs.
). Synapsin I cross-links synaptic vesicles and cytoskeleton, and the interactions of the protein with actin filaments and synaptic vesicles are regulated by phosphorylation by calmodulin-dependent protein kinase II (CaM kinase II)
). To understand the regulatory mechanisms of synapsin I function, it is necessary to know the posttranslational modifications of the protein in detail.
Recently we have applied electrospray mass spectrometry to studies on in vivo posttranslational modifications of various phosphoproteins. The high precision (within a few Da) and the high resolution (on the order of 10 Da) achieved by the method has made it possible to analyze protein phosphorylation and myristoylation of isolated proteins directly (
). Liquid chromatography/electrospray mass spectrometry (LC/MS), in which a capillary high performance liquid chromatography is connected online to an electrospray mass spectrometer, was found very useful in analyzing the in vivo posttranslational modifications including protein phosphorylation. Application of the methodology to brain-specific phosphoproteins revealed that prominent in vivo substrate proteins such as myristoylated alanine-rich protein kinase C substrate (MARCKS) or GAP-43 are phosphorylated by proline-directed protein kinases such as mitogen-activated protein (MAP) kinase and Cdk5 (
). This was surprising, since these two proteins have been believed to be major and specific substrates of protein kinase C. This prompted us to reexamine in vivo phosphorylation sites of various major phosphoproteins systematically.
In the present study, the posttranslational modifications of synapsin I isolated from bovine brain were studied, and the LC/MS analysis revealed a novel phosphorylation site. To identify the protein kinase(s) involved in the phosphorylation at the novel site, we have examined in vitro phosphorylation of synapsin I by three proline-directed protein kinases expressed highly in the brain, namely MAP kinase, Cdk5-p23 (tau protein kinase II), and glycogen synthase kinase 3β (GSK3β) (tau protein kinase I) (
). Effects of the phosphorylation on physiological functions of synapsin I were further assessed by examining the interaction of the protein with cytoskeletal proteins.
Synapsin I, one of the prominent endogenous phosphoproteins in the nerve terminals, has been characterized as a substrate protein of various protein kinases such as CaM kinases I and II and cAMP-dependent protein kinase (
). The detailed analysis on the in vivo phosphorylation site described in the present study, however, revealed a novel phosphorylation site. The phosphorylated serine is immediately followed by a proline, suggesting that synapsin I is an in vivo substrate of so-called proline-directed protein kinase. We have previously shown that the two prominent in vivo substrate proteins of protein kinase C, MARCKS and GAP-43, are also phosphorylated by these kinases in vivo (
). These results suggest that the physiological functions of various proteins are regulated by multiple protein kinases in a very complex manner and that cross-talks between various signaling pathways occur not only upstream of the pathways but also at the substrate protein level.
Of the three proline-directed protein kinases tested, only MAP kinase and Cdk5-p23 phosphorylated synapsin I in vitro, and GSK3β did not phosphorylate the protein to a significant extent. The phosphorylation by the two kinases was site-specific; only one of two serine residues (Ser551 and Ser553) was phosphorylated by Cdk5-p23, while three serine residues were phosphorylated by MAP kinase. Since bovine synapsin I contains 11 Ser (The)-Pro motifs, there should be structural determinants other than the adjacent proline in the substrate recognition by the kinases. As for MAP kinase, Ser551 is within a well known recognition sequence of the kinase, Pro-Xaa-Ser-Pro, where Xaa is usually a small neutral amino acid (
). Since the other two sites have a proline either at −3-position or at −1-position, the presence of a proline preceding the phosphorylation site serine/threonine may be important for the recognition. It should be noted that peptide T4 (from Leu8 to Arg53), which was phosphorylated to some extents, contains a single Ser-Pro motif (Ser39), which is preceded by a proline. On the other hand, only one phosphopeptide was observed with synapsin I phosphorylated by Cdk5-p23. This kinase seems to phosphorylate both Ser551 and Ser553, but the phosphorylation at these two sites is mutually exclusive, suggesting that the incorporation of negative charges in the neighborhood changes the substrate specificity. Ser551 is preferentially phosphorylated, although the difference may not be significant. The recognition sequence of the kinase has yet to be defined, but an arginine at +3-position may be an important determinant.
Only one kinase, so-called proline-directed protein kinase has been so far reported to phosphorylate synapsin I in these regions (
). Since Cdc2 kinase is not expressed in the brain to a significant extent, this phosphorylation reaction lacks physiological relevance. However, it is of interest to note that the kinase also phosphorylates Ser551 preferentially (
). Whether the kinase phosphorylates Ser553 is not clear, because of the limitation of the technique used in determining the phosphorylation site. The radiosequencing employed in the study suffers from a massive carryover, which obscures the determination of successive phosphorylation sites. In any case, it is interesting that the three proline-directed protein kinases so far tested phosphorylate Ser551 exclusively or preferentially. Only Cdk5-p23 phosphorylates Ser553, but the kinase phosphorylates Ser551 as well, although only the former was found phosphorylated in vivo as has been shown in the present study. One possibility is that a protein kinase or kinases other than the ones tested are responsible for the phosphorylation of Ser553. The other explanation is that Ser551 is preferentially dephosphorylated by protein phosphatase(s). Since the present study on the in vivo phosphorylation state of synapsin I represents only a “snapshot” of the total brain, similar studies conducted with cells and tissues under various physiological stimulations may give an answer to the question. The in vivo and in vitro phosphorylation sites so far identified are summarized in Fig. 8.
Phosphorylation of synapsin I at Ser568 and at Ser605 by CaM kinase II abolishes the bundling activity of actin filaments (
). As shown in the present study, phosphorylation by MAP kinase showed a similar effect on the F-actin bundling activity, while Cdk5-p23-dependent phosphorylation had practically no effect. Since MAP kinase and Cdk5-p23 both phosphorylate a similar site in the tail region of synapsin I (Ser551 or Ser553), the effects caused by the MAP kinase-dependent phosphorylation should be due to the phosphorylation of the two serine residues in the head region (Ser62 and Ser67). The major actin-binding site has been reported in the globular head domain of the synapsin I molecule (
). At the moment it is not clear whether the region around the two phosphorylation sites by MAP kinase is directly involved in the synapsin I-actin binding or if the conformational change caused by the phosphorylation is responsible for the diminished interaction. On the contrary, the binding of synapsin I to tubulin was not affected by the phosphorylation either by MAP kinase or Cdk5-p23. This may suggest that the binding sites for the two major cytoskeletal elements are different, and the interactions with them may be regulated differentially. Physiological function of the phosphorylation at the novel site found in the present study (Ser553), therefore, still remains to be established. Studies on the interaction of synapsin I with other cellular components such as synaptic vesicles and Grb2, a SH3-containing signal transduction protein (
Whether the MAP kinase- or Cdk5-p23-dependent phosphorylation occurs in vivo and whether the phosphorylation reactions are involved in the regulation of neurotransmitter release still remains to be seen. However, it should be noted that a mobility shift of synapsin I in SDS gel electrophoresis occurs in PC12 cells after nerve growth factor stimulation (
). According to these authors nerve growth factor induces a novel phosphorylation site of synapsin I at a site other than those by CaM kinase and by cAMP-dependent protein kinase. The phosphorylation has previously been attributed to that by Cdc2-cyclin A (
), but the present study demonstrated that the mobility shift is induced only by MAP kinase-dependent phosphorylation but not by Cdk5-p23-dependent phosphorylation. The fact that phosphorylation by Cdk5-p23 or that by Cdc2-cyclin A does not cause any detectable mobility shift (
) suggests that the protein kinase involved in nerve growth factor-dependent phosphorylation of synapsin I in PC12 is MAP kinase. In conclusion, proline-directed protein kinases that include MAP kinase, Cdk5-p23, and (probably) other unknown protein kinases seem to play important roles in the physiological regulation of cytoskeletal components during neurotransmitter release, and there seem to be complex interactions between various protein kinases not only upstream of the signal transduction pathways but also at the substrate protein level (