Rack1, a Receptor for Activated Protein Kinase C, Interacts with Integrin β Subunit

Abstract

The integrin ␤ subunit cytoplasmic domains are important for activation-dependent cell adhesion and adhesion-dependent signaling events. We report an interaction between integrin ␤ subunit cytoplasmic domain and Rack1, a Trp-Asp (WD) repeat protein that has been shown to bind activated protein kinase C. The Rack1-binding site on integrin ␤ 2 subunit resides within a conserved, membrane-proximal region. In the yeast twohybrid assay, WD repeats five to seven of Rack1 (Rack1-WD5/7) interact with integrin ␤ 1 , ␤ 2 , and ␤ 5 cytoplasmic domain. In eukaryotic cells, Rack1 co-immunoprecipitates with at least two different ␤ integrins, ␤ 1 integrins in 293T cells and ␤ 2 integrins in JY lymphoblastoid cells. Whereas Rack1-WD5/7 binds integrins constitutively, the association of full-length Rack1 to integrins in vivo requires a treatment with phorbol esters, which promotes cell spreading and adhesion. These findings suggest that Rack1 may link protein kinase C directly to integrins and participate in the regulation of integrin functions.Integrins are ␣␤ heterodimeric adhesion receptors that mediate attachment of cells to the extracellular matrix and specific cell counter-receptors (1). Various extracellular stimuli have been shown to affect the adhesiveness of integrins and regulate attachment of cells to the extracellular matrix (2). This process known as an activation-dependent cell adhesion is best illustrated in leukocytes where the attachment of integrin LFA1 1 (␣ L ␤ 2 ) to intercellular cell adhesion molecule-1 (ICAM-1) substrates can be promoted by cross-linking T cell receptors or stimulating cells with phorbol 12-myristate 13-acetate (PMA) (3). Upon binding to the extracellular matrix, integrins induce signals required for the reorganization of actin cytoskeleton and the formation of focal adhesion complexes (4 -6). The adhesion-dependent clustering of integrins leads to the activation of nonreceptor tyrosine kinase focal adhesion kinase and Ras/mitogen-activated protein kinase pathway, the stimulation of inositol lipid metabolism, an increase in intracellular Ca 2ϩ and pH, and the activation of PKC (4,7,8). Each subunit of integrins consists of a large extracellular ligand-binding domain, a transmembrane domain, and a short cytoplasmic domain that lacks any enzymatic activity. Although the cytoplasmic domains of ␣ subunits are variable in size and sequence, the cytoplasmic domains of ␤ subunits are more conserved in size and sequence. In particular, three conserved regions, termed cyto-1, cyto-2, and cyto-3, found in ␤ integrin cytoplasmic domains have been implicated in the recruitment of integrins to the focal adhesion plaques and the regulation of adhesive functions of integrins (9 -11). Although both integrin subunits are required for the ligand binding, the interaction between intracellular proteins and integrin cytoplasmic domain can occur in the absence of subunit association. Studies have shown that chimeric molecules composed of the ␤ integrin cytoplasmic domains and the extracellular domain of the i...

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