Journal: Journal of Biological Chemistry

Article Title: B Cell Receptor-induced Phosphorylation of Pyk2 and Focal Adhesion Kinase Involves Integrins and the Rap GTPases and Is Required for B Cell Spreading

doi: 10.1074/jbc.m109.013169

Figure Lengend Snippet: FIGURE 1. ExpressionandlocalizationofPyk2andFAKinBcells.A,Pyk2andFAKproteinlevelsincelllysates(40 gofprotein)fromA20Blymphomacells,restingsplenicBcells,andsplenicBcellsthatwereactivatedwithLPSplus IL-4 for 2 days were analyzed by sequential blotting with Abs to Pyk2, FAK, and Erk1/2 (loading control). Molecular massmarkers(inkDa)areindicatedtotherightofeachblot.TherelativeamountofPyk2orFAKproteininactivated BcellscomparedwithrestingBcells(1)wasdeterminedbyquantifyingbandintensitieswithImageJandnormal- izing the values to the amount of Erk in the sample. The data represent the mean S.E. for three independent experiments. B, the relative amounts of Pyk2 and FAK mRNA in resting and activated splenic B cells were determined by quantitative RT-PCR. Values were normalized to the amount of glyceraldehyde-3-phosphate dehydrogenasemRNAinthesamesampleandareexpressedastheamountofmRNA(averagerangefortwo independent experiments) relative to that in resting B cells (1). C, resting and LPS/IL-4-activated splenic B cells were left unstimulated for 30 min () or were incubated with 10 g/ml anti-IgM Abs for 30 min. Anti-Pyk2 immunoprecipitates (ippt) were analyzed by blotting with the 4G10 anti-Tyr(P) (P-Tyr) antibody (upper panel). The blots were then stripped and reprobed with an Ab to Pyk2 (lower panel). One of two experiments that yielded similar results is shown. D, RT-PCR analysis of alternatively spliced Pyk2 mRNA in resting splenic B cells and LPS/IL-4-activated B cells. PCR primers flanking the alternatively spliced exon distinguish the full-length mRNA from the spliced isoform, which is 126 bp shorter. Data are representative of three experiments with similar results. E, resting and LPS/IL-4-activated splenic B cells were permeabilized and stained with goat anti-Pyk2 or goat anti-FAK plus Alexa Fluor 488-conjugated secondary Ab. Nuclei were visualized with 4,6- diamidino-2-phenylindole (DAPI) (blue). No fluorescence was observed when the cells were stained with sec- ondary Ab alone or with nonspecific goat IgG plus secondary Ab (supplemental Fig. 1). F, LPS/IL-4 activated splenic B cells were permeabilized and stained with Abs to FAK plus Abs to either LFA-1 or VLA-4. In E and F, each panel shows representative data from one of three experiments with similar results.

Article Snippet: The rabbit polyclonal Ab against Tyr402-phosphorylated Pyk2, the murine monoclonal Ab against phosphorylated Erk, and the rabbit monoclonal Ab against Ser473-phosphorylated Akt were from Cell Signaling Technology (Danvers, MA).

Techniques: Control, Quantitative RT-PCR, Incubation, Reverse Transcription Polymerase Chain Reaction, Staining, Fluorescence

Journal: Journal of Biological Chemistry

Article Title: B Cell Receptor-induced Phosphorylation of Pyk2 and Focal Adhesion Kinase Involves Integrins and the Rap GTPases and Is Required for B Cell Spreading

doi: 10.1074/jbc.m109.013169

Figure Lengend Snippet: FIGURE 2. Adhesion of B cells to ECM selectively enhances BCR-induced tyrosine phosphorylation of Pyk2 and FAK. A20 cells were kept in suspen- sion or plated on collagen/fibronectin ECM for 30 min before being stimu- latedwith20g/mlsolubleanti-IgGfortheindicatedtimes.Forunstimulated controls (), A20 cells were kept in suspension or plated on collagen/fi- bronectin ECM for 30 min and then left unstimulated for an additional 45 min beforebeinglysed.A,immunoprecipitated(ippt)Pyk2andFAKwereanalyzed by immunoblotting with the 4G10 anti-Tyr(P) (P-Tyr) Ab. The blots were then reprobed with Abs to Pyk2 or FAK. A mock stimulation of cells with phos- phate-buffered saline for 15 or 30 min did not increase phosphorylation of Pyk2 and FAK compared with cells left unstimulated for the entire duration of the experiment (supplemental Fig. 2). B, cell lysates were immunoblotted with Abs against the phosphorylated forms of Erk (P-Erk) or Akt (P-Akt) and then reprobed with Abs against total Erk or Akt. C, cell lysates were immuno- blotted with a paxillin Ab. Serine/threonine phosphorylation of paxillin is indicatedbyabandshiftonSDS-PAGEgelsandwasdependentontheactivity of the Erk and GSK-3 kinases (data not shown) as in T cells and macrophages (67, 68). For each panel, similar results were obtained in three experiments. IgG, anti-IgG Ab.

Article Snippet: The rabbit polyclonal Ab against Tyr402-phosphorylated Pyk2, the murine monoclonal Ab against phosphorylated Erk, and the rabbit monoclonal Ab against Ser473-phosphorylated Akt were from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Suspension, Immunoprecipitation, Western Blot, Saline

Journal: Journal of Biological Chemistry

Article Title: B Cell Receptor-induced Phosphorylation of Pyk2 and Focal Adhesion Kinase Involves Integrins and the Rap GTPases and Is Required for B Cell Spreading

doi: 10.1074/jbc.m109.013169

Figure Lengend Snippet: FIGURE 4. BCR/integrin-induced tyrosine phosphorylation of Pyk2 and FAK depends on activation of the Rap GTPases. Vector control and RapGAPII-ex- pressing A20 cells were cultured for 30 min in wells coated with collagen/fi- bronectin ECM before being stimulated with 20 g/ml anti-IgG for the indicated times.Forunstimulatedcontrols(),A20cellswereplatedoncollagen/fibronec- tin ECM for 30 min and then left unstimulated for another 30 min before being lysed. A, anti-Pyk2 immunoprecipitates (ippt) were probed with an Ab against Pyk2 that is phosphorylated on Tyr402 (pY402) or with the 4G10 anti-Tyr(P) (P-Tyr) Abbeforebeingreprobedwithananti-Pyk2Ab.B,anti-FAKimmunoprecipitates were probed with an Ab that recognizes FAK that is phosphorylated on Tyr397 (pY397) or with the 4G10 anti-Tyr(P) Ab before being reprobed with an anti-FAK Ab. C, anti-FAK immunoprecipitates were probed sequentially with Abs that rec- ognize FAK that is phosphorylated on either Tyr576 or Tyr577 before being rep- robed with an anti-FAK Ab. The relative levels of Pyk2 and FAK phosphorylation were determined by quantifying band intensities using ImageJ, normalizing the values to the total amount of Pyk2 or FAK in the same lane, and expressing the values (mean S.E. for three experiments) relative to the Pyk2 or FAK phospho- rylation levels in unstimulated vector control cells (1). *, p 0.05 by Student’s one-tailed paired t test.

Article Snippet: The rabbit polyclonal Ab against Tyr402-phosphorylated Pyk2, the murine monoclonal Ab against phosphorylated Erk, and the rabbit monoclonal Ab against Ser473-phosphorylated Akt were from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Activation Assay, Plasmid Preparation, Control, Cell Culture, Expressing, One-tailed Test

Journal: Journal of Biological Chemistry

Article Title: B Cell Receptor-induced Phosphorylation of Pyk2 and Focal Adhesion Kinase Involves Integrins and the Rap GTPases and Is Required for B Cell Spreading

doi: 10.1074/jbc.m109.013169

Figure Lengend Snippet: FIGURE 5. Rap activation is important for integrin-induced phosphoryla- tion of Pyk2 and FAK. Vector control and RapGAPII-expressing A20 cells were plated in wells coated with 3.5 g/cm2 anti-CD40 (control), anti-LFA-1, or anti-VLA-4 monoclonal Abs for 15 or 30 min (). Anti-Pyk2 (A) and anti-FAK (B) immunoprecipitates (ippt) were analyzed by blotting with the 4G10 anti- Tyr(P) (P-Tyr) Ab. The blots were then stripped and reprobed with Abs against Pyk2 or FAK. For FAK phosphorylation, band intensities were normalized to the amount of total FAK for each sample and then expressed as the relative phosphorylation (mean S.E. for three experiments) compared with that for vector control cells plated on anti-CD40 (1). *, p 0.05 by Student’s one- tailed paired t test.

Article Snippet: The rabbit polyclonal Ab against Tyr402-phosphorylated Pyk2, the murine monoclonal Ab against phosphorylated Erk, and the rabbit monoclonal Ab against Ser473-phosphorylated Akt were from Cell Signaling Technology (Danvers, MA).

Techniques: Activation Assay, Plasmid Preparation, Control, Expressing, Phospho-proteomics, One-tailed Test

Journal: Journal of Biological Chemistry

Article Title: B Cell Receptor-induced Phosphorylation of Pyk2 and Focal Adhesion Kinase Involves Integrins and the Rap GTPases and Is Required for B Cell Spreading

doi: 10.1074/jbc.m109.013169

Figure Lengend Snippet: FIGURE 6. RapactivationisimportantforBCR-inducedtyrosinephosphorylationofPyk2butnotFAK.Vector control and RapGAPII-expressing A20 cells were stimulated in suspension with 20 g/ml anti-IgG for the indicated times. For unstimulated controls (), A20 cells were left in suspension for 30 min without being stimulated. A, anti- Pyk2 immunoprecipitates (ippt) were probed with either the 4G10 anti-Tyr(P) (P-Tyr) Ab, an Ab against Pyk2 that is phosphorylated on Tyr402, or an Ab against Pyk2 that is phosphorylated on Tyr579/Tyr580. The blots were then stripped and reprobed with a Pyk2 Ab. B, anti-FAK immunoprecipitates were probed with either the 4G10 anti-Tyr(P) Ab or an Ab against FAK that is phosphorylated on Tyr397. The blots were then stripped and rep- robed with a FAK Ab. Band intensities were normalized to the amount of total Pyk2 or FAK for each sample and then expressed as the relative phosphorylation (mean S.E. for three experiments) compared with that for unstimulated vector control cells (1). *, p 0.05 by Student’s one-tailed paired t test. The values for FAK phosphorylation in vector and RapGAPII-expressing cells were not significantly different by this test.

Article Snippet: The rabbit polyclonal Ab against Tyr402-phosphorylated Pyk2, the murine monoclonal Ab against phosphorylated Erk, and the rabbit monoclonal Ab against Ser473-phosphorylated Akt were from Cell Signaling Technology (Danvers, MA).

Techniques: Plasmid Preparation, Control, Expressing, Suspension, Phospho-proteomics, One-tailed Test

Journal: Journal of Biological Chemistry

Article Title: B Cell Receptor-induced Phosphorylation of Pyk2 and Focal Adhesion Kinase Involves Integrins and the Rap GTPases and Is Required for B Cell Spreading

doi: 10.1074/jbc.m109.013169

Figure Lengend Snippet: FIGURE 7. Rap-dependent phosphorylation of Pyk2 and FAK requires actin dynamics. A20 cells in suspension or plated on ECM were pretreated with 10 M latrunculin A or an equivalent volume of DMSO for 30 min before being stimulated with 20 g/ml soluble anti-IgG for the indicated times. For unstimulated controls (), A20 cells were kept in suspension or plated on collagen/fibronectin ECM for 30 min and then left unstimulated for another 30 min before being lysed. A, anti-Pyk2 immunoprecipitates (ippt) were sequentially probed with an Ab that recognizes Pyk2 that is phosphorylated at Tyr402, an Ab that recognizes Pyk2 that is phosphorylated at Tyr579/Tyr580, the 4G10 anti-Tyr(P) (P-Tyr) Ab, and an anti-Pyk2 Ab. B, cell lysates were assayed for phosphorylation of Akt (P-Akt) and Erk (P-Erk) as in Fig. 2B. C, a GST-RalGDS fusion protein was used to selectively precipitate the active GTP- bound form of Rap1, which was detected by immunoblotting with a Rap1 Ab (upper panel). The lower panel shows the amount of Rap1 in the cell lysates. D, tyrosine phosphorylation of FAK was assessed by blotting anti-FAK immu- noprecipitates with the 4G10 anti-Tyr(P) Ab and then reprobing with an anti-

Article Snippet: The rabbit polyclonal Ab against Tyr402-phosphorylated Pyk2, the murine monoclonal Ab against phosphorylated Erk, and the rabbit monoclonal Ab against Ser473-phosphorylated Akt were from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Suspension, Western Blot

Journal: Journal of Biological Chemistry

Article Title: B Cell Receptor-induced Phosphorylation of Pyk2 and Focal Adhesion Kinase Involves Integrins and the Rap GTPases and Is Required for B Cell Spreading

doi: 10.1074/jbc.m109.013169

Figure Lengend Snippet: FIGURE 8. An inhibitor of Pyk2/FAK activity blocks B cell spreading. A, A20 cells in suspension were treated with the indicated concentrations of PF-431396 or with DMSO for 45 min before being stimulated with 20 g/ml anti-IgG for 20 min. For unstimulated controls (), A20 cells were kept in suspension for 45 min and then left unstimulated for another 20 min before being lysed. Pyk2 and FAK immunoprecipitates (ippt) were ana- lyzed by blotting with the 4G10 anti-Tyr(P) (P-Tyr) Ab (left panel) before being reprobed with Abs against Pyk2 or FAK. The same cell lysates were analyzed for total tyrosine phosphorylation using the 4G10 anti-Tyr(P) Ab and for Erk phosphorylation (P-Erk) (right panel). B, A20 cells were treated with PF-431396 or DMSO for 45 min. The cells were then added to fibronectin/collagen ECM-coated wells and stimulated for 30 min with soluble anti-Ig. Alternatively the cells were added to wells coated with 2.63 g/cm2 anti-LFA-1 Abs for 30 min. Pyk2 and FAK immunoprecipitates were analyzed by blotting with the 4G10 anti-Tyr(P) Ab. C, A20 cells were plated on wells coated with 2.63 g/cm2 fibronectin (FN) and stimulated with 10 g/ml anti-IgG in the presence of DMSO or2.5MPF-431396(PF)for1,2,or4h.Representativeimagesofthe4-htimepointareshown.D,A20cellswere plated on wells coated with 2.63 g/cm2 anti-LFA-1 Ab in the presence of DMSO or 2.5 M PF-431396 for 1 or 3 h. Representative images of the 3-h time point are shown. Cell surface expression of LFA-1 was not affected by PF-431396 treatment (data not shown). The percentage of adherent A20 cells that had spread (mean S.E. for 150 cells counted in each of three experiments) as indicated by being phase dark with an elongated or irregular shape was determined for each time point. *, p 0.05 by Student’s one-tailed paired t test compared with DMSO-treated cells.

Article Snippet: The rabbit polyclonal Ab against Tyr402-phosphorylated Pyk2, the murine monoclonal Ab against phosphorylated Erk, and the rabbit monoclonal Ab against Ser473-phosphorylated Akt were from Cell Signaling Technology (Danvers, MA).

Techniques: Activity Assay, Suspension, Phospho-proteomics, Expressing, One-tailed Test

Journal: Arthritis Research & Therapy

Article Title: Differential expression of the FAK family kinases in rheumatoid arthritis and osteoarthritis synovial tissues

doi: 10.1186/ar2318

Figure Lengend Snippet: Rheumatoid arthritis synovial tissue (RA ST) had higher pPyk2 immunopositive cells compared to osteoarthritis (OA) ST. (a) RA (×200), compared to (b) OA (×200) and (c) normal donor (ND) (×200). (d) is the quantification data obtained from figure a and b. Bars represent mean and SEM. RA ST fibroblasts (e and f) or peripheral blood differentiated MΦs (g and h) were stimulated with TNF-α (10 ng/ml) or IL1-β (10 ng/ml) from 0–120 min. Cell lysates were examined by western blot analysis for pPyk2 or Pyk2 expression. The results are representative of three experiments. Inflam, inflammatory score; Vasc, vascularity score; Lining, ST lining cell layer; Mac, subsynovial MΦs.

Article Snippet: Blots were probed with rabbit anti-pFAK (Tyr 576/577), anti-pPyk2 (Tyr 402), or anti-pSrc (Tyr 527) (Cell Signaling Technology) overnight and after stripping reprobed with rabbit anti-FAK, anti-Pyk2 or anti-Src (Cell Signaling Technology at 1:1000) overnight.

Techniques: Western Blot, Expressing

Journal: Arthritis Research & Therapy

Article Title: Differential expression of the FAK family kinases in rheumatoid arthritis and osteoarthritis synovial tissues

doi: 10.1186/ar2318

Figure Lengend Snippet: Putative integrin signaling pathways through Pyk2 or FAK. In response to integrin αvβ3 activation, Pyk2 and/or FAK are recruited to a signaling complex that consists of Src, paxillin and PLCγ. Pyk2 may be phosphorylated through Src or other Ca 2+ dependent pathways whereas FAK is phosphorylated through Src. Both Pyk2 and FAK can result in activation of PI3K and/or MAPK that may lead to cell adhesion and migration into the rheumatoid arthritis synovial tissue (RA ST).

Article Snippet: Blots were probed with rabbit anti-pFAK (Tyr 576/577), anti-pPyk2 (Tyr 402), or anti-pSrc (Tyr 527) (Cell Signaling Technology) overnight and after stripping reprobed with rabbit anti-FAK, anti-Pyk2 or anti-Src (Cell Signaling Technology at 1:1000) overnight.

Techniques: Activation Assay, Migration