B cell receptor-induced phosphorylation of Pyk2 and focal adhesion kinase involves integrins and the Rap GTPases and is required for B cell spreading.
Signaling by the B cell receptor (BCR) promotes integrin-mediated adhesion and cytoskeletal reorganization. This results in B cell spreading, which enhances the ability of B cells to bind antigens and become activated. Proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK) are related cytoplasmic tyrosine kinases that regulate cell adhesion, cell morphology, and cell migration. In this report we show that BCR signaling and integrin signaling collaborate to induce the phosphorylation of Pyk2 and FAK on key tyrosine residues, a modification that increases the kinase activity of Pyk2 and FAK. Activation of the Rap GTPases is critical for BCR-induced integrin activation as well as for BCR- and integrin-induced reorganization of the actin cytoskeleton. We now show that Rap activation is essential for BCR-induced phosphorylation of Pyk2 and for integrin-induced phosphorylation of Pyk2 and FAK. Moreover Rap-dependent phosphorylation of Pyk2 and FAK required an intact actin cytoskeleton as well as actin dynamics, suggesting that Rap regulates Pyk2 and FAK via its effects on the actin cytoskeleton. Importantly B cell spreading induced by BCR/integrin co-stimulation or by integrin engagement was inhibited by short hairpin RNA-mediated knockdown of either Pyk2 or FAK expression and by treatment with PF-431396, a chemical inhibitor that blocks the kinase activities of both Pyk2 and FAK. Thus Pyk2 and FAK are downstream targets of the Rap GTPases that play a key role in regulating B cell morphology.
Tse
Kathy W K
KW
Department of Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Dang-Lawson
May
M
Lee
Rosaline L
RL
Vong
Doris
D
Bulic
Anica
A
Buckbinder
Leonard
L
Gold
Michael R
MR
D000199:Actins / Q000378:metabolism; D000818:Animals; D053265:B-Cell Activation Factor Receptor / Q000502:physiology*; D001402:B-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D051417:Focal Adhesion Kinase 1 / Q000235:genetics / Q000378:metabolism*; D051418:Focal Adhesion Kinase 2 / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D016023:Integrins / Q000037:antagonists & inhibitors / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D010766:Phosphorylation; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020668:rap GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*
D000199:Actins; D053265:B-Cell Activation Factor Receptor; D016023:Integrins; D034741:RNA, Small Interfering; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; C496051:Ptk2 protein, mouse; C496053:Ptk2b protein, mouse; D020668:rap GTP-Binding Proteins