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• W2049589697 abstract "SCG10 is a neuron-specific, membrane-associated protein that is highly concentrated in growth cones of developing neurons. Previous studies have suggested that it is a regulator of microtubule dynamics and that it may influence microtubule polymerization in growth cones. Here, we demonstrate that in vivo, SCG10 exists in both phosphorylated and unphosphorylated forms. By two-dimensional gel electrophoresis, two phosphoisoforms were detected in neonatal rat brain. Using in vitrophosphorylated recombinant protein, four phosphorylation sites were identified in the SCG10 sequence. Ser-50 and Ser-97 were the target sites for protein kinase A, Ser-62 and Ser-73 for mitogen-activated protein kinase and Ser-73 for cyclin-dependent kinase. We also show that overexpression of SCG10 induces a disruption of the microtubule network in COS-7 cells. By expressing different phosphorylation site mutants, we have dissected the roles of the individual phosphorylation sites in regulating its microtubule-destabilizing activity. We show that nonphosphorylatable mutants have increased activity, whereas mutants in which phosphorylation is mimicked by serine-to-aspartate substitutions have decreased activity. These data suggest that the microtubule-destabilizing activity of SCG10 is regulated by phosphorylation, and that SCG10 may link signal transduction of growth or guidance cues involving serine/threonine protein kinases to alterations of microtubule dynamics in the growth cone. SCG10 is a neuron-specific, membrane-associated protein that is highly concentrated in growth cones of developing neurons. Previous studies have suggested that it is a regulator of microtubule dynamics and that it may influence microtubule polymerization in growth cones. Here, we demonstrate that in vivo, SCG10 exists in both phosphorylated and unphosphorylated forms. By two-dimensional gel electrophoresis, two phosphoisoforms were detected in neonatal rat brain. Using in vitrophosphorylated recombinant protein, four phosphorylation sites were identified in the SCG10 sequence. Ser-50 and Ser-97 were the target sites for protein kinase A, Ser-62 and Ser-73 for mitogen-activated protein kinase and Ser-73 for cyclin-dependent kinase. We also show that overexpression of SCG10 induces a disruption of the microtubule network in COS-7 cells. By expressing different phosphorylation site mutants, we have dissected the roles of the individual phosphorylation sites in regulating its microtubule-destabilizing activity. We show that nonphosphorylatable mutants have increased activity, whereas mutants in which phosphorylation is mimicked by serine-to-aspartate substitutions have decreased activity. These data suggest that the microtubule-destabilizing activity of SCG10 is regulated by phosphorylation, and that SCG10 may link signal transduction of growth or guidance cues involving serine/threonine protein kinases to alterations of microtubule dynamics in the growth cone. SCG10 is a growth-associated protein abundant in the growth cones of developing neurons (1Stein R. Mori N. Matthews K. Lo L.C. Anderson D.J. Neuron. 1988; 1: 463-476Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 2Sugiura Y. Mori N. Dev. Brain Res. 1995; 90: 73-91Crossref PubMed Scopus (58) Google Scholar, 3Di Paolo G. Lutjens R. Osen-Sand A. Sobel A. Catsicas S. Grenningloh G. J. Neurosci. Res. 1997; 50: 1000-1009Crossref PubMed Scopus (57) Google Scholar). The gene encoding SCG10 is a member of the stathmin gene family (4Schubart U.K. Banerjee M. Eng J. DNA (N. Y.). 1989; 8: 389-398Crossref PubMed Scopus (69) Google Scholar). Both stathmin and SCG10 are microtubule (MT) 1The abbreviations used are: MT, microtubule; PKA, cAMP-dependent protein kinase; MAP, mitogen-activated protein; CDK (p34cdc2 and CDK5/p25), cyclin-dependent protein kinase; PAGE, polyacrylamide gel electrophoresis; NEPHGE, nonequilibrium pH gradient electrophoresis; HPLC, high performance liquid chromatography; MS, mass spectrometry; MS/MS tandem MS; LC, liquid chromatography; PBS, phosphate-buffered saline; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid. -destabilizing factors (5Belmont L.D. Mitchison T.J. Cell. 1996; 84: 623-631Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar, 6Marklund U. Larsson N. Melander Gradin H. Brattsand G. Gullberg M. EMBO J. 1996; 15: 5290-5298Crossref PubMed Scopus (248) Google Scholar, 7Riederer B.M. Pellier V. Antonsson B. Di Paolo G. Stimpson S.A. Lutjens R. Catsicas S. Grenningloh G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 741-745Crossref PubMed Scopus (168) Google Scholar). In in vitro assays of MT assembly, these molecules inhibit microtubule polymerization and induce depolymerization. Unlike stathmin, which is a cytosolic protein and expressed in most tissues (8Sobel A. Trends Biochem. Sci. 1991; 16: 301-305Abstract Full Text PDF PubMed Scopus (250) Google Scholar), SCG10 is a membrane-associated and neuron-specific protein (1Stein R. Mori N. Matthews K. Lo L.C. Anderson D.J. Neuron. 1988; 1: 463-476Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 9Di Paolo G. Lutjens R. Pellier V. Stimpson S.A. Beuchat M.-H. Catsicas S. Grenningloh G. J. Biol. Chem. 1997; 272: 5175-5182Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The expression of SCG10 is developmentally regulated, with high levels in embryonic and postnatal nervous system (1Stein R. Mori N. Matthews K. Lo L.C. Anderson D.J. Neuron. 1988; 1: 463-476Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 2Sugiura Y. Mori N. Dev. Brain Res. 1995; 90: 73-91Crossref PubMed Scopus (58) Google Scholar). In the adult, its expression persists in several brain regions that are associated with synaptic plasticity (10Himi T. Okazaki T. Wang H. McNeill T.H. Mori N. Neuroscience. 1994; 60: 907-926Crossref PubMed Scopus (65) Google Scholar), and up-regulation of SCG10 has been found following lesion experiments (11Mc Neill T.H. Cheng H.W. Rafols J.A. Mori N. Hefti F. Weiner W.J. Progress in Parkinson Research II. Plenum Press, New York1992: 299-323Google Scholar). Overexpression of SCG10 in a neuronal cell line was found to enhance neurite outgrowth (7Riederer B.M. Pellier V. Antonsson B. Di Paolo G. Stimpson S.A. Lutjens R. Catsicas S. Grenningloh G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 741-745Crossref PubMed Scopus (168) Google Scholar). Moreover, SCG10 is highly concentrated in the central domain of growth cones (3Di Paolo G. Lutjens R. Osen-Sand A. Sobel A. Catsicas S. Grenningloh G. J. Neurosci. Res. 1997; 50: 1000-1009Crossref PubMed Scopus (57) Google Scholar) where the distal ends of MTs are in a dynamic state of growth and shrinkage (12Letourneau P.C. Ressler A.H. J. Cell Biol. 1984; 98: 1355-1362Crossref PubMed Scopus (161) Google Scholar, 13Lim S.S. Sammak P.J. Borisy G. J. Cell Biol. 1989; 109: 253-263Crossref PubMed Scopus (139) Google Scholar, 14Okabe S. Hirokawa N. Nature. 1990; 343: 479-482Crossref PubMed Scopus (150) Google Scholar). Thus, SCG10 may be a regulator of MT dynamic instability during neurite outgrowth and structural plasticity. While the role of MTs in growth cone motility and neurite elongation is well established (15Tanaka E. J. Cell Biol. 1995; 115: 381-395Google Scholar, 16Tanaka E. Ho T. Kirschner M.W. J. Cell Biol. 1995; 128: 136-155Google Scholar, 17Williamson T. Gordon-Weeks P.R. Schachner M. Taylor J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15221-15226Crossref PubMed Scopus (115) Google Scholar, 18Challacombe J.F. Snow D.M. Letourneau P.C. J. Neurosci. 1997; 17: 3085-3095Crossref PubMed Google Scholar), little is known about their regulation in response to the environment and the signaling pathways involved. An understanding of phosphorylation and dephosphorylation events regulating the activity of SCG10 may lead to important insights into the intracellular mechanisms that modulate growth cone motility. We have previously shown that recombinant SCG10 is an in vitro target for the serine/threonine protein kinases PKA, MAP kinase, and cyclin-dependent kinase (CDK) p34cdc2 (19Antonsson B. Lutjens R. Di Paolo G. Kassel D. Allet B. Bernard A. Catsicas S. Grenningloh G. Protein Expr. Purif. 1997; 9: 363-371Crossref PubMed Scopus (27) Google Scholar). Both MAP kinase and PKA are present in growth cones and associated with microtubules (20Morishima-Kawashima M. Kosik K.S. Mol. Biol. Cell. 1996; 7: 893-905Crossref PubMed Scopus (144) Google Scholar). The CDK p34cdc2 is not expressed in neurons, but another member of the CDK family, CDK5/p25, which is highly homologous to p34cdc2, has been identified in neurons (21Lew J. Wang J.H. Trends Biochem. Sci. 1995; 20: 33-37Abstract Full Text PDF PubMed Scopus (186) Google Scholar, 22Tang D. Lee K.Y. Qi Z. Matsuura I. Wang J.H. Biochem. Cell Biol. 1996; 74: 419-429Crossref PubMed Scopus (28) Google Scholar) and is localized in growth cones (23Pigino G. Paglini G. Ulloa L. Avila J. Caceres A. J. Cell Sci. 1997; 110: 257-270Crossref PubMed Google Scholar). Here, we demonstrate that SCG10 is phosphorylated in vivo and we have identified four phosphorylation sites in the recombinant protein using liquid chromatography/electrospray ionization mass spectrometry. To further elucidate the molecular mechanism of SCG10 function, we have analyzed the effect of phosphorylation on the activity of the protein in intact cells. Our findings suggest that SCG10 is a phosphoprotein in developing brain and that its MT-destabilizing activity is regulated by phosphorylation. A rabbit antibody directed against SCG10 (anti-SCG10-BR) was generated by injecting 80 μg of recombinant, NH2-terminal truncated SCG10 that had been phosphorylated with PKA, MAP kinase, and p34cdc2 (19Antonsson B. Lutjens R. Di Paolo G. Kassel D. Allet B. Bernard A. Catsicas S. Grenningloh G. Protein Expr. Purif. 1997; 9: 363-371Crossref PubMed Scopus (27) Google Scholar) in complete Freund's adjuvant. One month after the first injection, the animal was boosted every 2 weeks with 40 μg of antigen in incomplete Freund's adjuvant. Five days after the third boost, the animal was deeply anesthetized with Nembutal and bled by cardiac puncture. The anti-SCG10 serum was tested for specificity in Western blots. For immunofluorescence experiments, the previously described rabbit polyclonal antibody directed against SCG10 (9Di Paolo G. Lutjens R. Pellier V. Stimpson S.A. Beuchat M.-H. Catsicas S. Grenningloh G. J. Biol. Chem. 1997; 272: 5175-5182Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) and a mouse monoclonal antibody for α-tubulin (clone B-5-1-2, Sigma) were used. Wistar rats of different ages (birth, postnatal days (P) 0, 5, 10, 15, and 20) and adult rats (3 months) were prepared for biochemistry as described earlier (24Riederer B.M. Guadano-Ferraz A. Innocenti G.M. Dev. Brain Res. 1990; 56: 235-243Crossref PubMed Scopus (73) Google Scholar). Animals were deeply anesthetized (40 mg of sodium pentobarbital per kg body weight) and decapitated. The brains were removed, immediately frozen in liquid nitrogen, and kept at −80 °C until use. Proteins (50 μg/slot) were separated on 5–20% gradient SDS-polyacrylamide gel electrophoresis and either stained with Coomassie Blue or transferred to nitrocellulose sheets. Western blots were incubated with anti-SCG10-BR antiserum (1:3000) followed by a peroxidase-conjugated secondary antibody and developed with 4-chloro-1-naphthol (24Riederer B.M. Guadano-Ferraz A. Innocenti G.M. Dev. Brain Res. 1990; 56: 235-243Crossref PubMed Scopus (73) Google Scholar). For dephosphorylation, the brain of a P5 animal was homogenized in dephosphorylation buffer (50 mm Tris/HCl, pH 8.2, 135 mm NaCl, 0.1 mm EDTA, 1 mm4-(2-aminoethyl)benzenesulfonyl fluoride, 30 μg/ml each of leupeptin, antipain, and pepstatin, and 15 μg/ml E64), and incubated with and without 0.2 IU alkaline phosphatase (Boehringer Mannheim, molecular grade)/μg of protein overnight at 37 °C (25Riederer B.M. Innocenti G.M. Eur. J. Neurosci. 1991; 3: 1134-1145Crossref PubMed Scopus (43) Google Scholar, 26Riederer B.M. Binder L.I. Brain Res. Bull. 1994; 33: 155-161Crossref PubMed Scopus (31) Google Scholar). The reaction was stopped by adding electrophoresis sample buffer and boiling. Control homogenates were incubated with 1 mm 4-nitrophenyl phosphate and 50 mm sodium fluoride to inhibit phosphatase activity. Samples were separated on SDS-PAGE, blotted onto nitrocellulose, and immunostained. Because SCG10 could not be resolved by regular isoelectrofocusing, brain homogenates and phosphorylated forms of the recombinant NH2-terminal truncated SCG10 were separated in the first dimension by a nonequilibrium pH gradient (NEPHGE) (27O'Farrell P. Goodman H.M. O'Farrell P.H. Cell. 1977; 12: 1133-1142Abstract Full Text PDF PubMed Scopus (2584) Google Scholar). Protein samples were dissolved in sample buffer (9.5 m urea, 2% CHAPS, 1% DTT, 0.8% ampholine, pH 3–10, 1.2% ampholine, pH 7–9, and 0.4% ampholine, pH 9–11) at 10 μg of protein/μl. Each gel was loaded with 50 μg of brain homogenate, or 1 and 0.3 μg of recombinant SCG10 for Coomassie Blue staining and Western blot, respectively. The upper buffer chamber contained 0.1 mH3PO4, the lower 0.02 m NaOH. The polarity was the reverse of that used for isoelectrofocusing gels, with the cathode (+) at the top and the anode (−) at the bottom. After application of the sample, the proteins were separated for 2 h at 800 V, 4 °C. The gels were incubated for 30 min with equilibration buffer (3% SDS, 1 mm EDTA, 10% glycerol, 0.2% 2-mercaptoethanol, 125 mm Tris-HCl, pH 6.8), and separated on a 5–20% acrylamide gradient (or 15% SDS-PAGE). Proteins were visualized by Coomassie Blue staining or after transfer to nitrocellulose by immunostaining. Recombinant SCG10 was purified as described earlier and stored in 20 mm Tris-HCl, 0.2 mm DTT, pH 7,5 at −80 °C (19Antonsson B. Lutjens R. Di Paolo G. Kassel D. Allet B. Bernard A. Catsicas S. Grenningloh G. Protein Expr. Purif. 1997; 9: 363-371Crossref PubMed Scopus (27) Google Scholar, 28Antonsson B. Montessuit S. Di Paolo G. Lutjens R. Grenningloh G. Protein Expr. Purif. 1997; 9: 295-300Crossref PubMed Scopus (12) Google Scholar). The purified protein was over 98% pure on reverse phase-HPLC and showed apparent homogeneity on SDS-PAGE. For the mass spectrometry studies, full-length SCG10 (19Antonsson B. Lutjens R. Di Paolo G. Kassel D. Allet B. Bernard A. Catsicas S. Grenningloh G. Protein Expr. Purif. 1997; 9: 363-371Crossref PubMed Scopus (27) Google Scholar) was used, whereas for the two-dimensional gel analysis, NH2-terminal truncated SCG10 (28Antonsson B. Montessuit S. Di Paolo G. Lutjens R. Grenningloh G. Protein Expr. Purif. 1997; 9: 295-300Crossref PubMed Scopus (12) Google Scholar) was used. For each phosphorylation assay, 250 pmol (5 μg) of the protein were used in a total reaction volume of 50 μl in the buffers described below. MAP kinase (p44mpk, Upstate Biotechnology Inc., Lake Placid, NY): 15 ng MAP kinase in 15 mm MOPS, pH 7.0, 10 mmMgCl2, 0.5 mm EGTA, 50 mm NaF, and 1 mm DTT. cAMP-dependent protein kinase, catalytic subunit (PKA, Boehringer Mannheim, Germany): 5 milliunits of PKA in 20 mm MOPS, pH 7.0, 10 mmMgCl2, 0.5 mm EGTA, and 1 mm DTT. p34cdc2 kinase (Promega, Madison, WI): 10 units pp34cdc2 in 20 mm MOPS, pH 7.0, 10 mmMgCl2, 0.5 mm EGTA, 50 mm NaF, and 0.5 mm DTT. All reaction mixtures contained 0.2 mm ATP. The samples were incubated at 35 °C for 60 min. The reactions were stopped by addition of 20 mm EDTA and the samples were stored at −80 °C until analyzed. Electrospray ionization-mass spectrometry (MS) was performed using an API-III triple quadrupole mass spectrometer (PE-Sciex, Concord, Ontario, Canada) equipped with an HP1090 microbore HPLC ternary pump system (Hewlett Packard, Palo Alto, CA). Separations were carried out using a PorosTM R2/H 300 mm × 10-cm capillary perfusion column (LC Packings, San Francisco, CA). Buffer A was 0.05% trifluoroacetic acid in H2O. Buffer B was 0.035% trifluoroacetic acid in 90/10 acetonitrile/H2O. The column flow rate was 50 μl/min and was achieved using a pre-column flow split. An aliquot of purified SCG10, corresponding to 50–100 pmol, was loaded onto the capillary perfusion column and eluted using a gradient of 15% buffer B to 65% buffer B in 5 min. The mass spectrometer was scanned from m/z 825 and m/z 1125 Da every 3 s during the gradient HPLC separation. The resolution of the mass spectrometer unit was up to m/z 2200 (20% valley definition) as determined from the infusion of a (poly)propylene glycol standard calibrant solution (supplied by PE-Sciex). Mass spectra were acquired using a 0.2-Da step size (permitting 5 data points/Da) and a 2.0-ms dwell time per step. Aliquots of protein samples, corresponding to 5–25 pmol, were injected onto an immobilized trypsin perfusion column (PerSeptive Biosystems, Framingham, MA) and digested on-column (total on-column digestion time of 2 min). Peptide fragments were trapped onto a reversed phase C18 column and eluted into the ion source of the mass spectrometer as described previously (29Lombardo C.R. Consler T.G. Kassel D.B. Biochemistry. 1995; 34: 16456-16466Crossref PubMed Scopus (53) Google Scholar). Tryptic peptides were separated on a 15 cm × 300-μm capillary C18 column (LC Packings) operated at 5 μl/min using a linear gradient of 1 to 21% buffer B in 5 min and 21 to 41% buffer B in 15 min. Electrospray mass spectra were acquired by scanning the mass spectrometer from 300–1800 Da in 3 s using a 0.5-Da step and a 1.0-ms dwell time. Alternatively, an aliquot of SCG10 corresponding to 25 pmol, was purified by reversed phase perfusion column HPLC and subjected to digestion with endoprotease Glu-C (Boehringer Mannheim, Germany) in NH4HCO3 at pH 8.0 for 12 h and at an enzyme:substrate ratio of 1:50 to confirm the sites of phosphorylation identified from the tryptic digestions. Phosphopeptides were identified from the tryptic and Glu-C digests using the stepped orifice voltage technique, previously reported by Huddleston et al.(30Huddleston M.J. Annan R. Bean M. Carr S.A. J. Am. Soc. Mass Spectrom. 1993; 4: 710-717Crossref PubMed Scopus (234) Google Scholar) and Ding et al. (31Ding J.M. Burkhart W. Kassel D.B. Rapid Commun. Mass Spectrom. 1994; 8: 94-98Crossref PubMed Scopus (83) Google Scholar). An aliquot of protein digest (5–25 pmol) was separated on the capillary C18 column and analyzed by electrospray ionization-MS. Peptides were ionized in the negative ion mode, and phosphopeptides were identified based on their ability to form a prominent PO3− ion, indicative of phosphorylation, at m/z 79. Tryptic phosphopeptides identified by negative ion stepped orifice potential experiments were subjected to on-line LC/MS/MS. These peptides were separated using the same column and gradient as described above. LC/MS/MS spectra of phosphopeptides were acquired by scanning the mass spectrometer from 50 Da to the mass of the precursor (M + H)+ ion in 3 s. The collision gas was set to 3 × 1014 collision gas thickness units. The mass spectrometer resolution was set to 1000 (full-width, half-maximum). The size of some of the tryptic fragments precluded their ability to be characterized fully by LC/MS/MS. Consequently, some of these larger tryptic fragments were subjected to digestion with Glu-C. These peptides were characterized by LC/MS/MS in a manner identical to the tryptic peptides, described above. The rat SCG10 cDNA was a generous gift of Dr. N. Mori (Kyoto, Japan). To replace serine with alanine or aspartate at positions 50, 62, 73, and 97 in the SCG10 sequence, site-directed mutagenesis was performed according to polymerase chain reaction procedures as described previously (32Nelson R.M. Long G.L. Anal. Biochem. 1989; 180: 147-151Crossref PubMed Scopus (294) Google Scholar). All mutants were subcloned into the BamHI and XbaI sites of the pcDNA3 expression vector (Invitrogen) and constructs were confirmed by DNA sequencing. COS-7 cells were cultured and electroporated as described previously (9Di Paolo G. Lutjens R. Pellier V. Stimpson S.A. Beuchat M.-H. Catsicas S. Grenningloh G. J. Biol. Chem. 1997; 272: 5175-5182Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) and plated in 100-mm Petri dishes containing 15-mm coverslips. After 48 h, cells were fixed for 20 min in 4% formaldehyde in phosphate-buffered saline (PBS) and washed 3 times with PBS. Cells were incubated for 2 h at room temperature with a rabbit antiserum to SCG10 (9Di Paolo G. Lutjens R. Pellier V. Stimpson S.A. Beuchat M.-H. Catsicas S. Grenningloh G. J. Biol. Chem. 1997; 272: 5175-5182Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) and mouse monoclonal antibodies to α-tubulin (clone B5-1-2, Sigma) in PBS containing 10% normal goat serum (Sigma), 0.3% Triton X-100, and 2% bovine serum albumin (Sigma). Following three washes with PBS, cells were incubated for 30 min at room temperature with fluorescein-conjugated goat anti-mouse and Cy3-conjugated goat anti-rabbit antibodies (Jackson Laboratories, Bar Harbor, ME). Cells were washed three times with PBS, and coverslips were mounted using Vectashield mounting medium (Vector). Using a fluorescent microscope (Zeiss Axioscop), cells expressing high levels of SCG10, i.e. where SCG10 staining was not restricted to the Golgi area, were assessed for their microtubule content. Cells were considered as cells containing polymerized MT if they contained more than 50 intact MTs. In three independent experiments, 50 cells were scored, and the percentage of cells containing polymerized MTs was calculated. As controls, we analyzed untransfected cells and cells that were transfected with an inactive SCG10 construct where the first 99 amino acids were deleted. In both cases 100% of the interphase cells showed a dense MT network. To compare the activity of SCG10 in transfected cells with that of a soluble form of SCG10 missing the membrane-binding domain (9Di Paolo G. Lutjens R. Pellier V. Stimpson S.A. Beuchat M.-H. Catsicas S. Grenningloh G. J. Biol. Chem. 1997; 272: 5175-5182Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), 100 transfected cells from three independent experiments were counted regardless of their level of expression, and the percentage of cells containing polymerized MTs was determined as described above. An anti-SCG10 serum, raised against in vitro phosphorylated recombinant SCG10 recognized two major bands in Western blots of postnatal brain extracts, one at 22 kDa and one at 25 kDa (Fig.1 A). As expected, due to the developmental change of SCG10 protein in rat brain (2Sugiura Y. Mori N. Dev. Brain Res. 1995; 90: 73-91Crossref PubMed Scopus (58) Google Scholar), the levels of expression decreased after postnatal day 5 and became undetectable in the adult (Fig. 1 A). Following alkaline phosphatase treatment of P5 brain extract, the band at 25 kDa was no longer detectable, indicating that it corresponds to one or several phosphorylated forms of SCG10 (Fig. 1 B). Two-dimensional gel electrophoresis revealed three isoforms of SCG10 in postnatal brain extract (Fig. 1 C). The electrophoretic mobility of these isoforms was compared with that of in vitro phosphorylated, NH2-terminal truncated SCG10 (28Antonsson B. Montessuit S. Di Paolo G. Lutjens R. Grenningloh G. Protein Expr. Purif. 1997; 9: 295-300Crossref PubMed Scopus (12) Google Scholar) (as described under “Experimental Procedures”) (Fig. 1, D–F). A mixture of nonphosphorylated SCG10 and SCG10 that was phosphorylated by CDK or by a combination of CDK, MAP kinase, and PKA generated a SCG10 pattern similar to that found in brain extracts (Fig. 1 G). These results indicate that postnatal rat brain contains unphosphorylated as well as two different phosphoisoforms of SCG10. For determination of the phosphorylation sites, recombinant SCG10 was in vitrophosphorylated by PKA, MAP kinase, and CDK as described under “Experimental Procedures.” The phosphorylated samples were first analyzed by ion-spray mass spectrometry to determine the number of phosphorylation sites. Ion-spray mass spectrometry gave a mass of 20,624 ± 1.4 for unphosphorylated SCG10, which is in agreement with the calculated molecular mass (20,624 Da). After phosphorylation with PKA, the main molecular mass was found to be 20,803, which corresponds to diphosphorylated SCG10. MAP kinase phosphorylation gave both mono- and diphosphorylated SCG10 with molecular masses of 20,715 and 20,803, respectively. However, after 120 min of incubation with the kinase, the protein was entirely diphosphorylated (Fig.2 A). Samples phosphorylated with CDK showed mainly monophosphorylated protein with a molecular mass of 20,715. To map the sites of phosphorylation, the samples were digested with either trypsin or Glu-C, and the peptides were separated on reverse phase-HPLC and sequenced with tandem mass spectrometry (MS/MS). After trypsin digestion of the sample phophorylated by PKA, three peptides were identified by the stepped orifice voltage technique and with a shift in the molecular masses on LC/MS. One peptide gave a (M + 3H)3+ signal at m/z 815.5 which corresponds to Mr 2,447 (Fig.3 A). The calculatedMr for the monophosphorylated peptide 48–69 is 2,445. When the peptide was sequenced, serine 50 was identified as the site of phosphorylation. The second peak corresponded to the peptide 49–69 and confirmed the phosphorylation site as serine 50. The third shifted peak was at a (M + 2H)2+ signal of m/z632.8 corresponding to a molecular mass of 1,266 (Fig. 3 B). The monophosphorylated peptide 95–104 has a calculatedMr = 1,266. MS/MS sequencing of the peptide revealed that the phosphorylation site was serine 97. The phosphorylation sites were confirmed by sequencing of two phosphorylated peptides from a Glu-C digestion (amino acid 42–55 and 93–99). In the samples phosphorylated with MAP kinase and digested with trypsin two phosphorylated peptides were identified and found to be shifted in the LC/MS spectrum (Table I). When the first peptide showing a (M + H)+ ion at m/z 696 was sequenced by MS/MS, the phosphorylation site was identified as serine 73. The second peptide gave a (M + 2H)2+ signalm/z 1145, which corresponds to a molecular mass of 2,290. In the SCG10 sequence this corresponds to a monophosphorylated peptide generated through cleavage at amino acid 49 and 69 (Mr 2,289). Sequencing of the peptide revealed that serine 62 was the site of phosphorylation (Fig. 2 B). Digestion with Glu-C also generated two phosphorylated peptides (amino acid 67–83 and 56–66). Sequencing of the peptides confirmed the phosphorylation sites.Table ISummary of data obtained for in vitro phosphorylated SCG10MassMass of shifted peptidesAmino acidsPeptide sequenceP-Ser residueDaDaSCG1020624SCG10 + PKA20803244748–69RApSGQAFELILKPPSPISEAPR50126695–104RKpSQEAQVLK97SCG10 + MAP kinase2071569670–75TLApSPK7320803229049–69ASGQAFELILKPPpSPISEAPR62SCG10 + CDK2071569670–75TLApSPK73 Open table in a new tab Phosphorylation with CDK resulted in one phosphorylated peptide with a (M + H)+ signal at m/z 695.5. MS/MS sequencing showed that the peptide corresponded to amino acid 70–75 with serine 73 phosphorylated (calculated Mr 696). The results are summarized in Table I and Fig.4, A and B, and compared with the phosphorylation sites of stathmin. SCG10 inhibits the assembly of microtubules and induces their disassembly in vitro (7Riederer B.M. Pellier V. Antonsson B. Di Paolo G. Stimpson S.A. Lutjens R. Catsicas S. Grenningloh G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 741-745Crossref PubMed Scopus (168) Google Scholar). To study the effect of SCG10 on microtubules in intact cells and to identify the role of phosphorylation in its activity, we have transiently transfected COS-7 cells, which do not express endogenous SCG10. We assessed the effects of expression of wild-type SCG10 on the MT array by immunofluorescence staining using an anti-α-tubulin antibody. Then, we tested mutants in which the serines, individually or in combination, were mutated to alanine or aspartate to prevent or mimic phosphorylation, respectively (Fig. 4 C). In cells expressing low levels of wild-type SCG10 in which SCG10 staining was observed in the area of the Golgi apparatus, as previously shown (9Di Paolo G. Lutjens R. Pellier V. Stimpson S.A. Beuchat M.-H. Catsicas S. Grenningloh G. J. Biol. Chem. 1997; 272: 5175-5182Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), no obvious abnormalities could be observed. These cells were similar to the controls where 100% of the interphase cells showed a dense MT network. However, cells expressing high levels of SCG10, where the protein was also abundant in the cytoplasm (Fig.5 B), showed a dramatic, sometimes complete, disappearance of the microtubule network (Fig. 5,A and B). When we compared the activity of wild-type SCG10 with that of a construct that is missing the NH2-terminal membrane-binding domain and thus expressed as a cytosolic protein (9Di Paolo G. Lutjens R. Pellier V. Stimpson S.A. Beuchat M.-H. Catsicas S. Grenningloh G. J. Biol. Chem. 1997; 272: 5175-5182Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), we found that SCG10 is less active in the membrane-bound form. While 43.7 ± 1.2% of cells transfected with full-length SCG10, only 20.3 ± 0.9% of cells transfected with soluble SCG10 contained polymerized MTs (p < 0.001). We compared the effects of high level expression of wild-type SCG10 with the effects of SCG10 in which serine-to-alanine substitutions were made at each individual site (S50A, S62A, S73A, and S97A) or at dual sites (S50A,S97A, S62A,S73A). Analysis showed no statistically significant difference in depolymerizing activity between these proteins (Fig. 6). However, the nonphosphorylatable mutant (S50A,S62A,S73A,S97A) was significantly more active in depolymerizing the microtubule network than the wild-type protein (Figs. 5, C and D, and 6). The percentage of cells containing polymerized microtubules decreased from 13.3 ± 0.9% for wild-type to 3.0 ± 1.5% for the nonphosphorylatable mutant (p < 0.001) (Fig. 6). To further test the role of phosphorylation in regulating the MT destabilizing activity of SCG10, mutants in which serine residues were substituted with aspartate residues at one, two, or all four sites were analyzed (Fig. 4 C). While introducing acidic charges to mimic phosphorylation at either Ser-50 or Ser-62 had no significant effect on the activity of SCG10, single mutations at Ser-73 (S73D) and Ser-97 (S97D) significantly decreased SCG10 activity (Fig. 6). The percentage of cells containing polymerized microtubules increased from 13.3 ± 0.9% for wild-type to 24.0 ± 0.6% and 27.0 ± 1.5%, respectively, for these mutants (p < 0.001). Double phosphorylation at Ser-50 and Ser-97 (S50D,S97D) and at Ser-62 and Ser-73 (S62D,S73D) showed a further increase in cells containing microtubules to 31.7 ± 2.8% and 28.7 ± 1.3, respectively (p < 0.001). The greatest reduction in SCG10 activity was observed when all four phosphorylation sites were substituted by aspartate (S50D, S62D, S73D, and S97D) resulting in an increase of cells containing MTs to 46.7 ± 0.9% (p < 0.001) (Fig. 6). To assess whether endogenous phosphorylation of SCG10 in COS-7 cells contributes to reducing its activity, the double aspartate mutant S62D,S73D was further mutated on Ser-50 and Ser-97 with alanine substitutions to prevent phosphorylation on these two residues (S50A,S97A,S62D,S73D). The percentage of cells transfected with S50A,S97A,S62D,S73D which contained polymerized MTs (25.3 ± 1.8%) was similar to that of cells transfected with S62D,S73D (28.7 ± 1.3%), indicating that the endogenous phosphorylation on Ser-50 and Ser-97 in this mutant does not contribute significantly to decreasing SCG10 activity (Fig. 6). Altogether, our results suggest that phosphorylation negatively regulates the microtubule-depolymerizing activity of SCG10 and that all four sites participate in this regulation, although the relative importance of each site varies. SCG10, a growth cone-enriched MT-destabilizing protein, has been recently characterized as an in vitro substrate for various serine/threonine kinases including PKA, MAP kinase, and CDK (19Antonsson B. Lutjens R. Di Paolo G. Kassel D. Allet B. Bernard A. Catsicas S. Grenningloh G. Protein Expr. Purif. 1997; 9: 363-371Crossref PubMed Scopus (27) Google Scholar). We have found that SCG10 is phosphorylated in vivo in developing rat brain. The in vivo isoforms correspond to unphosphorylated, monophosphorylated, and multiphosphorylated forms. More detailed studies will be required to reveal the specific phosphorylation states of this protein in cells and tissues under a variety of physiological conditions. In this work, the sites of SCG10 phosphorylated by PKA, MAP kinase, and CDK have been identified. Our results, based upon tryptic peptide mapping followed by LC/MS and MS/MS sequencing, are summarized in Fig.4 A, where they are compared with the sites that have been reported for the related protein stathmin (33Beretta L. Dobransky T. Sobel A. J. Biol. Chem. 1993; 268: 20076-20084Abstract Full Text PDF PubMed Google Scholar, 34Leighton I.A. Curmi P. Cambell D.G. Cohen P. Sobel A. Mol. Cell. Biochem. 1993; 127–128: 151-156Crossref PubMed Scopus (69) Google Scholar, 35Marklund U. Brattsand G. Shingler V. Gullberg M. J. Biol. Chem. 1993; 268: 15039-15047Abstract Full Text PDF PubMed Google Scholar). The PKA phosphorylation sites present in SCG10 are conserved to the in vitro and in vivo phosphorylation sites known for stathmin. The sites for MAP kinase phosphorylation were identified as Ser-62 and Ser-73 of SCG10. Both sites contain a proline residue C-terminal to the serine consistent with the serine (threonine)-proline specificity of this kinase (Fig. 4 B). In contrast to stathmin, where the major site for MAP kinase was Ser-25, no preferred phosphorylation site was found for SCG10. Two sites (Ser-25 and Ser-38) are phosphorylated by CDK in stathmin, whereas we found only one major site in SCG10 that was phosphorylated by this kinase (Ser-73). Since the kinase used for these experiments is not expressed in neurons, the neuronal cdc2-like kinase CDK5/p25, which was also found to efficiently phosphorylate SCG10 (data not shown), may be the physiologically relevant kinase. Our results suggest that the function of SCG10 is regulated by multiple protein kinases and that the kinases PKA, MAP kinase, and CDK5/p25, all three of which are present in growth cones (20Morishima-Kawashima M. Kosik K.S. Mol. Biol. Cell. 1996; 7: 893-905Crossref PubMed Scopus (144) Google Scholar, 23Pigino G. Paglini G. Ulloa L. Avila J. Caceres A. J. Cell Sci. 1997; 110: 257-270Crossref PubMed Google Scholar), are good candidates to phosphorylate SCG10 in vivo. To reveal physiological functions of SCG10 phosphorylation, we assessed whether phosphorylation at the identified sites had an effect on the MT-destabilizing activity of the protein. We found that overexpression of wild-type SCG10 in COS-7 cells caused disruption of the MT network consistent with its microtubule-depolymerizing effect in vitro (7Riederer B.M. Pellier V. Antonsson B. Di Paolo G. Stimpson S.A. Lutjens R. Catsicas S. Grenningloh G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 741-745Crossref PubMed Scopus (168) Google Scholar). A similar activity was recently reported for the cytosolic protein stathmin upon transfection (6Marklund U. Larsson N. Melander Gradin H. Brattsand G. Gullberg M. EMBO J. 1996; 15: 5290-5298Crossref PubMed Scopus (248) Google Scholar) or microinjection of recombinant protein (36Horwitz S.B. Shen H.-J. He L. Dittmar P. Neef R. Chen J. Schubart U.K. J. Biol. Chem. 1997; 272: 8129-8132Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) into cells. However, SCG10 was significantly less active than a truncated cytosolic form of the protein, and its effect was observed mainly in highly overexpressing cells, where SCG10 localization was not restricted to the area of the Golgi complex (9Di Paolo G. Lutjens R. Pellier V. Stimpson S.A. Beuchat M.-H. Catsicas S. Grenningloh G. J. Biol. Chem. 1997; 272: 5175-5182Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) but also found in the cytoplasm. This may indicate that Golgi-associated SCG10 is either not very active or, more likely, not in a subcellular compartment where it can induce depolymerization of interphase microtubules. It is not known yet whether SCG10 functions while it is bound to organelles in neuronal growth cones (3Di Paolo G. Lutjens R. Osen-Sand A. Sobel A. Catsicas S. Grenningloh G. J. Neurosci. Res. 1997; 50: 1000-1009Crossref PubMed Scopus (57) Google Scholar) or whether it has to be released from membranes. By expressing a series of phosphorylation site mutants, we showed that the MT-destabilizing effect of SCG10 could be modulated. While the nonphosphorylatable mutant showed higher activity than the wild-type protein, the activity of the mutant in which phosphorylation on all four sites was mimicked by an aspartate residue was greatly reduced. These data suggest that the nonphosphorylated state of SCG10 represents the most active form of the protein. Observations of stathmin in transfected or microinjected cells (6Marklund U. Larsson N. Melander Gradin H. Brattsand G. Gullberg M. EMBO J. 1996; 15: 5290-5298Crossref PubMed Scopus (248) Google Scholar, 36Horwitz S.B. Shen H.-J. He L. Dittmar P. Neef R. Chen J. Schubart U.K. J. Biol. Chem. 1997; 272: 8129-8132Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) as well as in an in vitro assay of MT assembly (37Larsson N. Marklund U. Gradin H.M. Brattsand G. Gullberg M Mol. Cell. Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (169) Google Scholar, 38Di Paolo G. Antonsson B. Kassel D. Riederer B.M. Grenningloh G. FEBS Lett. 1997; 416: 149-152Crossref PubMed Scopus (78) Google Scholar) suggest a similar mechanism of regulation of the activity of the two proteins. However, for stathmin it has been reported that alanine substitution on only two of the four serines (Ser-25 and Ser-38) increased its MT-destabilizing activity to nearly the same extend as mutating all four sites. This was not the case for SCG10, where the S50A,S62A,S73A,S97A mutant was significantly more active than any single or double alanine substitutions. Interestingly, these two serine residues in stathmin (Ser-25 and Ser-38) are not precisely conserved in the SCG10 sequence (Fig.4 B) and exhibit differences between stathmin and SCG10 in their phosphorylation by MAP kinase and CDK (Fig. 4 A). We also tested the effect of mutation of phosphorylation sites in SCG10 by introducing aspartate residues to replace each of the four phosphorylatable serines, both individually and in various combinations. Of the single mutants, only S73D and S97D were statistically different from wild-type, but not from each other. Also the double mutants were not statistically different from each other, though they were different from the S50D,S62D,S73D,S97D mutant. Whether the decreased activity of aspartate mutants is caused by a reduced binding to tubulin as is the case for in vitrophosphorylated stathmin (37Larsson N. Marklund U. Gradin H.M. Brattsand G. Gullberg M Mol. Cell. Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (169) Google Scholar, 38Di Paolo G. Antonsson B. Kassel D. Riederer B.M. Grenningloh G. FEBS Lett. 1997; 416: 149-152Crossref PubMed Scopus (78) Google Scholar) needs to be determined. In summary, our results strongly suggest that the activity of SCG10 is controlled by phosphorylation and that its activity can be down-regulated to different extents by multiple phosphorylations. Therefore, SCG10 may be a key factor that links growth or guidance cues to the local control of MT assembly in growth cones. Fine tuning of its activity, possibly by several signal transduction pathways that act in concert, may be involved in regulation of the dynamics of MTs required for growth cone advance and turning. We thank all members of the neurobiology group at Geneva Biomedical Research Institute for their helpful discussion. We are grateful to R. Golsteyn for experiments on SCG10 phosphorylation with neuronal CDK. We also thank A. Bernard, H. Blasey, J. Y. Bonnefoy, N. Gullu, C. Hebert, S. Herren, P. Graber, S. Montessuit, R. Porchet, L. Potier, and E. Sebille for their help at various stages of this work. We thank S. Catsicas and J.K. Staple for many helpful comments and critical reading the manuscript." @default.
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• W2049589697 title "Identification of in Vitro Phosphorylation Sites in the Growth Cone Protein SCG10" @default.
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