p38 mitogen-activated protein kinase interacts with vinculin at focal adhesions during fatty acid-stimulated cell adhesion.
Arachidonic acid stimulates cell adhesion by activating α2β1 integrins in a process that depends on protein kinases, including p38 mitogen activated protein kinase. Here, we describe the interaction of cytoskeletal components with key signaling molecules that contribute to the spreading of, and morphological changes in, arachidonic acid-treated MDA-MB-435 human breast carcinoma cells. Arachidonic acid-treated cells showed increased attachment and spreading on collagen type IV, as measured by electric cell-substrate impedance sensing. Fatty acid-treated cells displayed short cortical actin filaments associated with an increased number of β1 integrin-containing pseudopodia, whereas untreated cells displayed elongated stress fibers and fewer clusters of β1 integrins. Confocal microscopy of arachidonic acid-treated cells showed that vinculin and phospho-p38 both appeared enriched in pseudopodia and at the tips of actin filaments, and fluorescence ratio imaging indicated the increase was specific for the phospho-(active) form of p38. Immunoprecipitates of phospho-p38 from extracts of arachidonic acid-treated cells contained vinculin, and GST-vinculin fusion proteins carrying the central region of vinculin bound phospho-p38, whereas fusion proteins expressing the terminal portions of vinculin did not. These data suggest that phospho-p38 associates with particular domains on critical focal adhesion proteins that are involved in tumor cell adhesion and spreading, and that this association can be regulated by factors in the tumor microenvironment.
National Institute of Environmental Health Sciences, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA.
Margaret D
MD
George
Robert N
RN
Wine
Brad
B
Lackford
Grace E
GE
Kissling
Steven K
SK
Akiyama
Kenneth
K
Olden
John D
JD
Roberts
D016718:Arachidonic Acid / Q000494:pharmacology*; D001665:Binding Sites; D002448:Cell Adhesion / Q000187:drug effects; D045744:Cell Line, Tumor; D024141:Collagen Type IV / Q000235:genetics / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000235:genetics* / Q000648:ultrastructure; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D019012:Integrin beta1 / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011554:Pseudopodia / Q000187:drug effects / Q000235:genetics* / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D016596:Vinculin / Q000235:genetics / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*
D024141:Collagen Type IV; D019012:Integrin beta1; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C488955:VCL protein, human; D016596:Vinculin; D016718:Arachidonic Acid; D048051:p38 Mitogen-Activated Protein Kinases