Direct transmembrane clustering and cytoplasmic dimerization of focal adhesion kinase initiates its tyrosine phosphorylation.
We investigated mechanisms for inducing focal adhesion kinase (FAK) tyrosine phosphorylation and their ability to trigger MAP kinase signaling using transmembrane chimeras that localize FAK and its mutants to the plasma membrane. We tested whether tyrosine phosphorylation was triggered by FAK transmembrane aggregation using antibodies against the chimeric extracellular domain. Experimental clustering of chimeras containing integrin beta cytoplasmic domains or FAK induced FAK tyrosine phosphorylation and trans-phosphorylation of endogenous FAK, as well as strong ERK activation. Next, we examined whether lower-order molecular proximity, namely dimerization, could regulate FAK tyrosine phosphorylation. We found that even relatively low-affinity FAK dimerization (K(d)=3.9 x 10(-5) M), in either of two different orientations, could induce FAK tyrosine phosphorylation. However, this cytoplasmic FAK dimerization could not induce MAP kinase activation or trans-phosphorylation of endogenous FAK. We conclude that dimerization of FAK is sufficient to induce its tyrosine phosphorylation, but that higher-order molecular proximity (clustering) at the cell membrane is apparently needed for additional biochemical events. This study identifies a proximity mechanism for regulating the initiation of FAK-mediated biochemical signaling.
Katz
Ben-Zion
BZ
Craniofacial Developmental Biology and Regeneration Branch, National Institutes of Health, Bethesda, MD 20892-4370, USA. sbkatz@netvision.net.il
Miyamoto
Shingo
S
Teramoto
Hidemi
H
Zohar
Muriel
M
Krylov
Dmitry
D
Vinson
Charles
C
Gutkind
J Silvio
JS
Yamada
Kenneth M
KM
D016475:3T3 Cells; D000818:Animals; D002448:Cell Adhesion; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism; D019281:Dimerization; D004789:Enzyme Activation; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D016350:Leucine Zippers; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; D020928:Mitogen-Activated Protein Kinases