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 i nitiates signaling events regulating integrin-mediated cell adhesion. CD2 stimulation of resting human T cells results within minutes in an increase in beta 1-integrin-mediated adhesion to fibronectin. We have utilized the HL60 cell line to map critical residues within the CD2 cy toplasmic domain involved in CD2 regulation of integrin function. A pa nel 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 i nterleukin-2 production or CD2 avidity do not affect CD2 regulation of integrin activity. A proline-rich sequence, K-G-PP-L-P (amino acids 2 99 to 305), is essential for CD2-mediated regulation of pi integrin ac tivity. CD2-induced increases in beta 1 integrin activity could be blo cked by two phosphoinositide 3-kinase (PI 3-K) inhibitors or by overex pression of a dominant negative form of the p85 subunit of PI 3-K. In addition, CD2 cytoplasmic domain mutations that abrogate CD2-induced i ncreases in integrin-mediated adhesion also ablate CD2-induced increas es in PI 3-K enzymatic activity. Surprisingly, CD2 cytoplasmic domain mutations that inhibit CD2 regulation of adhesion do not affect the co nstitutive association of the p85 subunit of PI 3-K association with C D2. Mutation of the proline residues in the K-G-P-P-L-P motif to alani nes prevented CD2-mediated activation of integrin function and PI 3-K activity but not mitogen-activated protein (MAP) kinase activity. Furt hermore, the MEK inhibitor PD 098059 blocked CD2-mediated activation o f 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 (beta 1 integrin adhesion by CD2. In addition, these stu dies suggest that CD2-mediated activation of MAP kinase is not involve d in CD2 regulation of integrin adhesion.