Impaired integrin-mediated signal transduction, altered cytoskeletal structure and reduced motility in Hck/Fgr deficient macrophages.
Integrin-mediated adhesion of monocytes and macrophages initiates a signal transduction pathway that leads to actin cytoskeletal reorganization, cell migration and immunologic activation. This signaling pathway is critically dependent on tyrosine kinases. To investigate the role of the Src-family of tyrosine kinases in integrin signal transduction, we have examined the adhesive properties of macrophages isolated from hck-/-fgr-/- double knockout mice which lack two of the three predominant Src-family kinases expressed in myeloid cells. Previous examination of polymorphonuclear leukocytes from these animals indicated that these kinases were critical in initiating the actin cytoskeletal rearrangements that lead to respiratory burst and granule secretion following integrin ligation. Double mutant peritoneal exudate macrophages demonstrated markedly reduced tyrosine phosphorylation responses compared to wild-type cells following plating on fibronectin, collagen or vitronectin-coated surfaces. Tyrosine phosphorylation of several actin-associated proteins (cortactin, paxillin, and tensin), as well as the Syk and Pyk2 tyrosine kinases, were all significantly reduced in double mutant cells. The subcellular localization of focal-adhesion associated proteins was also dramatically altered in mutant macrophages cultured on fibronectin-coated surfaces. In wild-type cells, filamentous actin, paxillin, and talin were concentrated along leading edges of the plasma membrane, suggesting that these proteins contribute to cellular polarization during migration in culture. Double mutant cells failed to show the polarized subcellular localization of these proteins. Likewise, double mutant macrophages failed to form normal filopodia under standard culture conditions. Together, these signaling and cytoskeletal defects may contribute to the reduced motility observed in in vitro assays. These data provide biochemical and morphological evidence that the Src-family kinases Hck and Fgr are required for normal integrin-mediated signal transduction in murine macrophages.
Suen
P W
PW
Departments of Laboratory Medicine, and Stomatology and Anatomy, University of California San Francisco, San Francisco, CA 94143, USA.
Ilic
D
D
Caveggion
E
E
Berton
G
G
Damsky
C H
CH
Lowell
C A
CA
D000199:Actins / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D015703:Antigens, CD / Q000737:chemistry / Q000276:immunology; D001854:Bone Marrow Cells / Q000166:cytology; D002364:Caseins / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000187:drug effects / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000494:pharmacology; D005353:Fibronectins / Q000494:pharmacology; D051418:Focal Adhesion Kinase 2; D039482:Integrin alpha5; D019012:Integrin beta1 / Q000737:chemistry / Q000276:immunology; D016023:Integrins / Q000378:metabolism / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D017737:Macrophages, Peritoneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000172:deficiency* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D051572:Proto-Oncogene Proteins c-hck; D011554:Pseudopodia; D015398:Signal Transduction; D000072377:Syk Kinase; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases
D000199:Actins; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D002364:Caseins; D015815:Cell Adhesion Molecules; D003432:Cross-Linking Reagents; D004792:Enzyme Precursors; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D039482:Integrin alpha5; D019012:Integrin beta1; D016023:Integrins; D047908:Intracellular Signaling Peptides and Proteins; D011518:Proto-Oncogene Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; C496632:Hck protein, mouse; D051572:Proto-Oncogene Proteins c-hck; C496053:Ptk2b protein, mouse; D000072377:Syk Kinase; C000606897:Syk protein, mouse; C053102:proto-oncogene proteins c-fgr; D019061:src-Family Kinases