Canonical pathways that are significantly represented by the identified phosphoproteins. 1775 of 1883 identified phosphoproteins were mapped to the Ingenuity Pathways Analysis data base and used for pathway analysis. 78 pathways have a p value lower than 0.05 as determined by Fisher's test. Shown are 28 of the most significant pathways. ILK, Integrin linked kinase; PAK, p21-activated kinase; AMPK, AMP-activated protein kinase; HGF, hepatocyte growth factor; GnRH, gonadotropin-releasing hormone.

ERK kinase activation mediates NIE-115 neurite extension. A, neurons were allowed to extend neurites through 3.0-μm pores toward laminin for 12 h (growth) before LPA was added to the lower chamber for the indicated times to induce growth cone collapse and retraction. Proteins from somas or neurites were then specifically isolated as described under “Experimental Procedures” and Western-blotted with phosphosite-specific antibodies that recognize the phosphorylated activated form of ERK (P-ERK) or MEK (Thr-218, Thr-222, human sequence) or phosphoamino acids Ser-298 and Ser-292 of MEK, which modulate kinase activity in response to PAK and ERK kinase activity, respectively. Western blots of total cell ERK/MEK protein are shown for comparison. B, neurites were allowed to extend toward laminin for 18 h (time 0) then induced to collapse with LPA for the indicated times and Western-blotted for activated ERK-P and total ERK as described in A. C, soma and neurite proteins were prepared as in A and Western-blotted for the phosphorylated/activated form of Akt (p-Akt, Ser-473) or total Akt protein. D, cells were allowed to extend neurites for 18 h (growth) and then stimulated with LPA to induce retraction. Rac and Cdc42 activation (Rac-GTP, Cdc42-GTP) were determined from isolated neurite and soma proteins using a GST-PBD pulldown assay and Western blot analyses. ERK served as the loading control. E, cells were allowed to extend neurites to the lower surface for 8 h (growth) and then stimulated with LPA for the indicated times. Somas and neurites were purified and Western-blotted for the indicated proteins and/or specific phosphorylation sites. Crk Tyr-221 (PY) phosphorylation was detected as a decrease in electrophoretic mobility in the gel. LASP-1 was first immunoprecipitated (IP) and then Western-blotted with anti-phosphotyrosine antibodies. The asterisk indicates the neurite/soma fold enrichment of the protein as determined by mass spectrometry and or Western blotting and densitometry. F, cells were allowed to extend neurites to the lower surface for 8 h (growth) and then stimulated with LPA for the indicated times. Somas and neurites were purified and Western-blotted with anti-phosphotyrosine antibodies. Arrows show neurite-specific proteins that decrease in response to LPA. The asterisk shows a neurite protein band that does not change in response to LPA. G, N1E-115 cells were either held in suspension (S) or allowed to attach (A) to laminin-coated dishes for 45 min in the presence or absence of LPA and then Western-blotted for activated ERK (p-ERK) or MEK Ser-298 as in A.

Inhibition of ROCK kinase activity prevents LPA-induce N1E-115 neurite retraction and prevents ERK inactivation. A, neurites were allowed to extend to the lower surface of the membrane for 18 h and then treated with or without the ROCK kinase inhibitor Y-27632 for 30 min before being stimulated with or without LPA for 10 min. Neurites were quantified as described in Fig. 1D. B, neurites were isolated from cells treated as in A and Western-blotted for activated ERK (p-ERK) or phosphorylated myosin light chain (MLC-P) on serine 19 or total MLC. C, a schematic model shows the possible signaling and phosphorylation mechanisms that mediate neurite extension and retraction in response to the collapsing factor LPA. Gray-shaded circles with black text indicate the phosphorylation site and neurite growth/retraction ratio determined by mass spectrometry. Red text indicates -fold change in protein abundance level between soma and neurite. A list of the depicted proteins and their associated regulatory molecules and their neurite abundance and phosphorylation status is shown in supplemental Table 3. a shows integrin-mediated ERK activation and the neurite extension pathway. b shows LPA the induced neurite retraction pathway. Also see supplemental Fig. 1 for more details of the LPA/Rho pathway. c shows cytoskeleton-associated proteins that contain at least one phosphorylation site that conforms to the ERK amino acid consensus sequence PX(S)TP where X is any basic or neutral amino acid. Numbers in parentheses indicate the neurite growth/retraction ratio of the predicated ERK phosphorylation site as measured by mass spectrometry. MT, microtubules, MAPS, microtubule-associated proteins; LPAR, LPA receptor. PRKCZ, Protein kinase C ζ; PRKACG, cAMP-dependent protein kinase subunit γ; LCK, lymphocyte protein tyrosine kinase; NES, Nuclear Exclusion sequence; EBD, ERK binding domain and KD, Kinase Domain.

Characterization of neurite growth and retraction kinetics using 3.0-μm porous filters and the collapsing factor LPA. A, shown is a schematic of a neurite purification chamber showing neurite extension toward the lower surface of the filter coated with laminin. B, left panel, a fluorescence photomicrograph of the lower surface of the membrane shows neurites stained with α-tubulin (red) extending through 3.0-μm pores toward a drop of laminin (green) for 18 h. Note that neurites do not extend to the lower surface in the absence of laminin coating. Right panel, shown is a fluorescence photomicrograph of a NIE-115 neurite growth cone stained with FITC-phalloidin to visualize F-actin (green) and antibodies to tubulin to visualize microtubules (red). Bar, left panel, 30 μm; right panel, 10 μm. C, shown is quantification of neurite extension kinetics to the lower surface of 3.0-μm membranes coated with laminin on the bottom only, top only, bottom and top, or not coated with laminin. Neurites were stained with crystal violet; the dye was eluted and quantified using a spectrophotometer as described under “Experimental Procedures.” S.D. from three independent experiments are shown. D, shown is quantification of neurite retraction kinetics in response to LPA added to the lower chamber (left graph), upper or lower chamber (middle graph), or upper and lower chamber (right graph) for the indicated times. E, shown is a fluorescence photomicrograph of a time series of neurite retraction on the lower membrane surface in response to LPA added to the lower chamber for the indicated times. The arrow shows retracting neurites. Bar = 20 μm.

NetworKIN annotation of kinase substrates in growing and retracting neurites. The identified phosphosites in growing and retracting neurites were assigned to their cognate kinase class using the NetworkIN software program. The normalized ratio of the number of substrates for each kinase was calculated, and only those that have more than two substrates and with the ratio >1.5 or <0.67 were shown for comparison. PKACA, cAMP-dependent protein kinase catalytic subunit α; MET, hepatocyte growth factor receptor MET; BTK, Bruton tyrosine kinase; KIT, KIT kinase; INSR, insulin receptor; ITK, ITK tyrosine kinase; SGK, serine/threonine-protein kinase Sgk.

Deactivation of ERK is necessary for LPA-induced N1E-115 neurite retraction. A, quantification is shown of neurite formation of NIE-115 cells either mock-transfected or transfected with constructs encoding mutationally activated MEK (MEK-CA) or MEK-CA with Ser-298 or Ser-292 amino acids changed to alanine. Inset shows the level of MEK expression by Western blotting. B, shown is quantification of neurite formation for 6 h in the presence of the MEK kinase inhibitor PD98059 (25 μm) or the vehicle DMSO (control). The inset shows the level of ERK phosphorylation and kinase activity by Western blotting as described in Fig. 4A. C, cells were either mock-transfected with an empty vector or transfected with HA-tagged MEK-CA and allowed to extend neurites to the lower surface for 18 h and then treated with or without LPA for 10 min to induce retraction and quantified as described in Fig. 1D. D, somas and neurites were purified from cells transfected as in C and Western-blotted with phosphospecific antibodies to activated p-ERK and MEK phosphorylated on Ser-292 or Ser-298. HA-MEK and total ERK Western blots are shown for comparison. E, cells were allowed to extend neurites to the lower membrane surface for 12 h then treated with the MEK inhibitor PD98059 for 60 min (time 0) before being treated with or without LPA for the indicated times. Neurites were quantified as in Fig. 1D.