Identification of a proline-rich sequence in the CD2 cytoplasmic domain critical for regulation of integrin-mediated adhesion and activation of phosphoinositide 3-kinase.
The CD2 molecule is one of several lymphocyte receptors that rapidly initiates signaling events regulating integrin-mediated cell adhesion. CD2 stimulation of resting human T cells results within minutes in an increase in beta1-integrin-mediated adhesion to fibronectin. We have utilized the HL60 cell line to map critical residues within the CD2 cytoplasmic domain involved in CD2 regulation of integrin function. A panel of CD2 cytoplasmic domain mutants was constructed and analyzed for their ability to upregulate integrin-mediated adhesion to fibronectin. Mutations in the CD2 cytoplasmic domain implicated in CD2-mediated interleukin-2 production or CD2 avidity do not affect CD2 regulation of integrin activity. A proline-rich sequence, K-G-P-P-L-P (amino acids 299 to 305), is essential for CD2-mediated regulation of beta1 integrin activity. CD2-induced increases in beta1 integrin activity could be blocked by two phosphoinositide 3-kinase (PI 3-K) inhibitors or by overexpression of a dominant negative form of the p85 subunit of PI 3-K. In addition, CD2 cytoplasmic domain mutations that abrogate CD2-induced increases in integrin-mediated adhesion also ablate CD2-induced increases in PI 3-K enzymatic activity. Surprisingly, CD2 cytoplasmic domain mutations that inhibit CD2 regulation of adhesion do not affect the constitutive association of the p85 subunit of PI 3-K association with CD2. Mutation of the proline residues in the K-G-P-P-L-P motif to alanines prevented CD2-mediated activation of integrin function and PI 3-K activity but not mitogen-activated protein (MAP) kinase activity. Furthermore, the MEK inhibitor PD 098059 blocked CD2-mediated activation of MAP kinase but had no effect on CD2-induced adhesion. These studies identify a proline-rich sequence in CD2 critical for PI 3-K-dependent regulation of beta1 integrin adhesion by CD2. In addition, these studies suggest that CD2-mediated activation of MAP kinase is not involved in CD2 regulation of integrin adhesion.
Kivens
W J
WJ
Department of Laboratory Medicine and Pathology, Center for Immunology, and Cancer Center, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Hunt
S W
SW
Mobley
J L
JL
Zell
T
T
Dell
C L
CL
Bierer
B E
BE
Shimizu
Y
Y
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology; D018801:CD2 Antigens / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D003593:Cytoplasm; D004789:Enzyme Activation; D005353:Fibronectins / Q000502:physiology; D005982:Glutathione Transferase / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D019012:Integrin beta1 / Q000502:physiology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011392:Proline; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D013601:T-Lymphocytes / Q000276:immunology / Q000502:physiology*; D014162:Transfection
D015703:Antigens, CD; D018801:CD2 Antigens; D005353:Fibronectins; D019012:Integrin beta1; D011993:Recombinant Fusion Proteins; D011392:Proline; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases