Regulation of beta 1-integrin-mediated cell adhesion by the Cbl adaptor protein.
BACKGROUND
Leukocyte activation results in a rapid increase in adhesion to the extracellular matrix due to the activation of beta 1 integrin receptors. A role for phosphatidylinositol (PI) 3-kinase in integrin activation has been proposed, as activation of integrins by many receptors can be blocked by PI 3-kinase inhibitors. One receptor that regulates integrins is the CD28 surface antigen; here, we investigated the mechanisms responsible for CD28-mediated integrin activation.


RESULTS
CD28-mediated integrin activation was blocked by mutation of the binding site for the p85 catalytic subunit of PI 3-kinase in the CD28 cytoplasmic domain, and by expression of a dominant-negative form of the p85 subunit. Substitution of the Src homology 2 (SH2)-binding motif in the CD28 cytoplasmic domain for the corresponding motif in the CD28-related CTLA-4 surface antigen also blocked integrin activation but did not affect the recruitment and activation of PI 3-kinase. Mutations of the CD28 cytoplasmic domain that blocked integrin activation also impaired the tyrosine phosphorylation of the Cbl adaptor protein and the activation of the PI 3-kinase that was associated with Cbl. This Cbl-associated PI 3-kinase was distinct from the PI 3-kinase that coprecipitated with the CD28 cytoplasmic domain. CD28-mediated activation of beta 1 integrins was inhibited by expression of a mutant Cbl protein that shows reduced association with PI 3-kinase.


CONCLUSIONS
Cbl is required for PI-3-kinase-dependent regulation of integrin receptors by CD28. Furthermore, CD28 is coupled to two distinct pools of PI 3-kinase, one directly associated with the CD28 cytoplasmic tail and the other associated with Cbl.
Zell
T
T
Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis 55455, USA.
Warden
C S
CS
Chan
A S
AS
Cook
M E
ME
Dell
C L
CL
Hunt
S W
SW
Shimizu
Y
Y
D019943:Amino Acid Substitution; D015703:Antigens, CD / Q000096:biosynthesis / Q000502:physiology*; D001665:Binding Sites; D018801:CD2 Antigens / Q000096:biosynthesis / Q000502:physiology*; D018106:CD28 Antigens / Q000096:biosynthesis / Q000502:physiology*; D002448:Cell Adhesion / Q000502:physiology*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D018922:HL-60 Cells; D006801:Humans; D019012:Integrin beta1 / Q000502:physiology*; D007700:Kinetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D044767:Ubiquitin-Protein Ligases
D015703:Antigens, CD; D018801:CD2 Antigens; D018106:CD28 Antigens; D019012:Integrin beta1; D008164:Luminescent Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019000:Phosphotyrosine; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D019869:Phosphatidylinositol 3-Kinases; C067419:CBL protein, human; D013755:Tetradecanoylphorbol Acetate