Overrepresented GO biological process categories. LPA-regulated phosphoproteins from total cell lysate were compared with all phosphoproteins identified in the total cell lysate. Significantly overrepresented GO biological process terms (p < 0.05) are shown. Percentage values indicate the fractions of proteins annotated to the listed GO biological terms found in all LPA-regulated or all identified phosphoproteins with annotated GO biological process terms.

Differential phosphorylation changes upon treatment with LPA and HB-EGF. A scatter plot analysis of phosphorylation sites significantly and reproducibly changed upon LPA (●) or HB-EGF (▴) treatment in both experiments is shown. Values on the x axis (y axis) correspond to log₂-transformed average phosphorylation ratios determined upon HB-EGF (LPA) stimulation of A498 cells.

MS spectra from SILAC-based quantitative analysis. A, MS spectra of peptides derived from the tyrosine kinase Abl. The peptide with the amino acid sequence GQGESDPLDHEPAVSPLLPR was quantified in its non-phosphorylated and singly phosphorylated form harboring Ser(P)⁵⁸⁸. B, MS spectra of a set of peptides derived from the serine/threonine kinase PKD1. Peptides harboring the phosphorylation sites on Ser²⁰⁵ and Ser²⁰⁸ were quantified as non-, singly, or doubly phosphorylated forms with either phosphorylation detected in distinct singly modified peptide variants. A and B, isotopic variants originated from the differentially labeled and stimulated cells as indicated. Ratios obtained by SILAC-based quantification are shown. p(S), phosphoserine.

Stimulation conditions and experimental design. A, LPA- and HB-EGF-induced tyrosine phosphorylation of the EGFR in SILAC-encoded A498 cells. Immunoblot analysis of total cell extracts with antibody recognizing Tyr(P)¹¹⁷³ in the EGFR revealed similar stimulation upon treatment with either 0.5 ng/ml HB-EGF or 10 μm LPA. EGFR levels were similar as verified by immunoblotting with anti-EGFR antibody. B, schematic illustration of the integrated proteomics work flow for quantitative phosphorylation analysis of total lysates and kinase-enriched fractions. pY, phosphotyrosine.

Statistical and comparative analyses of quantitative MS data. Quantified phosphopeptides were binned according to their log₂ values of the ratio of their HB-EGF (A) or LPA (B) versus control ratios obtained in biological replicates. Gaussian regression analysis was applied to calculate the mean and S.D. for the log₂-transformed ratios of ratios. C, correlation of phosphorylation site ratios upon HB-EGF or LPA treatment shown in a scatter plot comparison of biological replicates. Consistently up- and down-regulated sites enclosed by either of the two rectangles were considered for further analysis. D, regulated phosphosite ratios determined from kinase-enriched fractions were similar with or without normalization for protein abundance as visualized by a scatter plot.

Network of LPA-regulated phosphoproteins. Known physical and functional interactions among proteins with LPA-regulated phosphorylation sites were visualized using the STRING program. The thickness of the edges provides a measure for the confidence of the reported interactions according to the combination of experimental and database evidence. Dashed edges indicate interactions, which have been manually added to the STRING-generated network based on literature evidence. Gene names are displayed in the network that correspond to the following proteins: ABL1, proto-oncogene tyrosine-protein kinase ABL1; ABL2, tyrosine-protein kinase ABL2; AHNAK, neuroblast differentiation-associated protein AHNAK; AKAP13, protein kinase A anchor protein 13; ANK2, Ankyrin-2; BAIAP2, brain-specific angiogenesis inhibitor 1-associated protein 2; BMP, bone morphogenetic protein 1; CAMKK1, calcium/calmodulin-dependent protein kinase kinase 1; CARHSP1, calcium-regulated heat stable protein 1; CAST, calcium-binding protein CAST; CAMKD, calcium/calmodulin-dependent protein kinase II δ; CAMK2G, calcium/calmodulin-dependent protein kinase type II γ chain; CDC25C, M phase inducer phosphatase 3; CD2AP, CD2-associated protein; CD44, CD44 antigen; CHEK1, serine/threonine-protein kinase Chk1; CLASP1, CLIP-associating protein 1; CLIP1, CAP-Gly domain-containing linker protein 1; CSNK1D, casein kinase I isoform δ; CXCR4, CXC chemokine receptor type 4; DDEF1, 130-kDa phosphatidylinositol 4,5-bisphosphate-dependent ARF1 GTPase-activating protein; DNM1, dynamin-1; EPHB2, ephrin type-B receptor 2; ERK1, mitogen-activated protein kinase 3; ERK2, mitogen-activated protein kinase 1; FAT, protocadherin FAT-1; F11R, junctional adhesion molecule A; GABRA4, γ-aminobutyric acid receptor subunit α-4; GNA12, guanine nucleotide-binding protein subunit α-12; GRDN, girdin; ITSN1, intersectin-1; MAP1B, microtubule-associated protein 1B; MARCKS, myristoylated alanine-rich protein kinase C substrate; MET, hepatocyte growth factor receptor; MEKK3, mitogen-activated protein kinase kinase kinase 3; PEA, astrocytic phosphoprotein PEA15; PLC3B, 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase β-3; PLEC, plectin-1; PRKD1, serine/threonine-protein kinase D1; PRKCG, protein kinase C γ type; PRKCH, protein kinase C η type; PRKRA, interferon-inducible double-stranded RNA-dependent protein kinase activator A; TK2B, protein-tyrosine kinase 2β; PTPN12, protein-tyrosine phosphatase non-receptor type 12; PTPN14, protein-tyrosine phosphatase non-receptor type 14; PXN, paxillin; RAI14, ankycorbin; RPS6KA1/3, ribosomal protein S6 kinase α-1/3; SPTBN1, spectrin β chain, brain 1; SRC, proto-oncogene tyrosine-protein kinase Src; TAOK1/2, serine/threonine-protein kinase TAO-1/2; TBC1D4, TBC1 domain family member 4; TEC, tyrosine-protein kinase TEC; TNIK, TRAF2 and NCK-interacting protein kinase; ZO1, tight junction protein ZO-1; ZYX, zyxin.