To investigate the molecular basis of PTENmediated tumor suppression, we introduced a null mutation into the mouse Pten gene by homologous recombination in embryonic stem (ES) cells. Pten ؊/؊ ES cells exhibited an increased growth rate and proliferated even in the absence of serum. ES cells lacking PTEN function also displayed advanced entry into S phase. This accelerated G 1 ͞S transition was accompanied by down-regulation of p27 KIP1 , a major inhibitor for G 1 cyclindependent kinases. Inactivation of PTEN in ES cells and in embryonic fibroblasts resulted in elevated levels of phosphatidylinositol 3,4,5,-trisphosphate, a product of phosphatidylinositol 3 kinase. Consequently, PTEN deficiency led to dosagedependent increases in phosphorylation and activation of Akt͞ protein kinase B, a well-characterized target of the phosphatidylinositol 3 kinase signaling pathway. Akt activation increased Bad phosphorylation and promoted Pten ؊/؊ cell survival. Our studies suggest that PTEN regulates the phosphatidylinositol 3,4,5,-trisphosphate and Akt signaling pathway and consequently modulates two critical cellular processes: cell cycle progression and cell survival.The tumor susceptibility gene encoding PTEN͞MMAC1͞TEP1 (1-3) is mutated at high frequency in many primary human cancers and several familial cancer predisposition disorders (4). PTEN contains the sequence motif that is highly conserved in the members of the protein tyrosine phosphatase family. PTEN has been shown in vitro to possess phosphatase activity on phosphotyrosyl, phosphothreonyl-containing substrates (3, 5) and more recently, on phosphatidylinositol 3,4,5-trisphosphate (PIP3), a product of phosphatidylinositol 3 (PI3) kinase (6). Many cancerrelated mutations have been mapped within the conserved catalytic domain of PTEN, suggesting that the phosphatase activity of PTEN is required for tumor suppressor function. In addition, wild-type PTEN, but not mutant derivatives lacking phosphatase activity, suppresses the growth of glioblastoma cells and their tumorigenecity in nude mice (7-9), confirming the functional relevance of the PTEN phosphatase domain for tumor suppression. Very recently, inactivation of PTEN in a mouse model has confirmed the role of PTEN as a bona fide tumor suppressor (10). However, the exact function of PTEN in regulation of cell growth and tumorigenesis remains unclear.In this study, we have investigated the molecular basis underlying the tumor suppression function of PTEN by using a combination of molecular genetic, cell biological, and biochemical approaches. We have identified PIP3, a product of PI3 kinase, as an intracellular target of PTEN. Our studies suggest that PTEN acts as a negative regulator for the PI3-kinase͞Akt signaling pathway, which controls and coordinates two major cellular processes: cell cycle progression and cell death.

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...

Pten (Phosphatase and tensin homolog deleted on chromosome 10) is a recently identified tumor suppressor gene which is deleted or mutated in a variety of primary human cancers and in three cancer predisposition syndromes [1]. Pten regulates apoptosis and cell cycle progression through its phosphatase activity on phosphatidylinositol (PI) 3,4,5-trisphosphate (PI(3,4,5)P(3)), a product of PI 3-kinase [2-5]. Pten has also been implicated in controlling cell migration [6], but the exact mechanism is not very clear. Using the isogenic Pten(+/+) and Pten(-/-) mouse fibroblast lines, here we show that Pten deficiency led to increased cell motility. Reintroducing the wild-type Pten, but not the catalytically inactive Pten C124S or lipid-phosphatase-deficient Pten G129E mutant, reduced the enhanced cell motility of Pten-deficient cells. Moreover, phosphorylation of the focal adhesion kinase p125(FAK) was not changed in Pten(-/-) cells. Instead, significant increases in the endogenous activities of Rac1 and Cdc42, two small GTPases involved in regulating the actin cytoskeleton [7], were observed in Pten(-/-) cells. Overexpression of dominant-negative mutant forms of Rac1 and Cdc42 reversed the cell migration phenotype of Pten(-/-) cells. Thus, our studies suggest that Pten negatively controls cell motility through its lipid phosphatase activity by down-regulating Rac1 and Cdc42.