Integrin-linked kinase, a promising cancer therapeutic target: biochemical and biological properties.

Integrin-linked kinase (ILK) is an ankyrin repeat-containing Ser/Thr kinase that interacts with the cytoplasmic domains of beta(1) and beta(3) integrins. ILK is widely expressed in tissues throughout the body, and, as might be expected, appears to mediate a diversity of functions relating to its role in coupling integrins and growth factor receptors to downstream signaling pathways. Through its downstream targets protein kinase B/Akt and glycogen synthase kinase-3beta, ILK appears to be involved in several oncogenesis-related events, including suppression of apoptosis and promotion of cell survival, as well as cell migration and invasion. Over-expression of ILK in epithelial cells results in anchorage-independent cell growth with increased cell cycle progression. Inoculation of nude mice with ILK over-expressing cells leads to tumor formation. Furthermore, increased ILK expression and activity have been correlated with malignancy in several human tumor types, including breast, prostate, brain, and colon carcinomas. Based on these findings, ILK represents an excellent therapeutic target for the prevention of tumor progression. Here, we provide an overview of the physical and biochemical properties of ILK, and present data describing the impact of small-molecule ILK inhibitors on several ILK-mediated cellular functions.

CD44-initiated cell spreading induces Pyk2 phosphorylation, is mediated by Src family kinases, and is negatively regulated by CD45.

CD44 is a cell adhesion molecule implicated in leukocyte adhesion and migration, co-stimulation of T cells, and tumor metastasis. CD45 is a leukocyte-specific protein tyrosine phosphatase that dephosphorylates the Src family kinases, Lck and Fyn, in T cells. Positive regulation of Lck by CD45 is required for its effective participation in T cell receptor signaling events. Here, immobilized CD44 antibody induced a distinctive cell spreading in CD45(-), but not CD45(+), T cells, and this correlated with the induction of tyrosine-phosphorylated proteins. Two focal adhesion family kinases, Pyk2 and, to a lesser extent, FAK were inducibly phosphorylated, as was a potential substrate, Cas. CD44-mediated cell spreading and induced tyrosine phosphorylation were prevented by the Src family kinase inhibitor, PP2. Furthermore, 2-fold more Lck associated with CD44 in the low density sucrose fraction from CD45(-) T cells compared with CD45(+) T cells, suggesting that CD45 may regulate the association of Lck with CD44 in this fraction. Therefore, in CD45(-) T cells, CD44 signaling is mediated by Src family kinases, and this leads to Pyk2 phosphorylation, cytoskeletal changes, and cell spreading. This implicates CD45 in the negative regulation of Src family kinase-mediated CD44 signaling leading to T cell spreading.

Integrin-linked kinase (ILK): a "hot" therapeutic target.

Integrin-mediated cell adhesion is known to regulate gene expression through the activation of transcription factors. We have recently revealed that these activations are mediated through integrin-linked kinase (ILK). ILK is an ankyrin repeat-containing serine-threonine protein kinase that can interact directly with the cytoplasmic domain of the beta1 and beta3 integrin subunits and whose kinase activity is modulated by cell-extracellular matrix interactions. We have shown that ILK overexpression results in the translocation of beta-catenin to the nucleus, which then forms a complex formation with the lymphoid enhancer binding factor 1 (LEF-1) transcription factor, subsequently activating the transcriptional activity of promoters containing LEF-1 response elements. ILK phosphorylates the glycogen synthase kinase-3 (GSK-3), which inhibits GSK-3 activity. We have demonstrated that ILK stimulates activator protein-1 transcriptional activity through GSK-3 and the subsequent regulation of the c-Jun-DNA interaction. ILK also phosphorylates protein kinase B (PKB/Akt) and stimulates its activity. We have shown that ILK is an upstream effector of the phosphatidylinositol 3-kinase-dependent regulation of PKB/Akt. ILK has been shown to phosphorylate PKB/Akt on Ser-473 in vitro and in vivo. Our results clearly indicate that ILK is a key element in the regulation of integrin signaling as well as growth factor and Wnt signaling pathways. PTEN (phosphatase and tensin homolog detected on chromosome 10) is a tumor suppressor gene located on chromosome 10q23 that encodes a protein and phospholipid phosphatase. It is now estimated that inactivation mutants of PTEN exist in 60% of all forms of solid tumors. Loss of expression or mutational inactivation of PTEN leads to the constitutive activation of PKB/Akt via enhanced phosphorylation of Thr-308 and Ser-473. We have demonstrated that the activity of ILK is constitutively elevated in PTEN mutant cells. A small molecule ILK inhibitor suppresses the phosphorylation of PKB at the Ser-473 but not the Thr-308 site in the PTEN mutant cells. These results indicate that inhibition of ILK may be of significant value in solid tumor therapy.

CD45 and RPTPalpha display different protein tyrosine phosphatase activities in T lymphocytes.

To examine the substrate specificity and function of two receptor protein tyrosine phosphatases, CD45 and RPTPalpha, RPTPalpha was expressed in a CD45(-), T-cell receptor (TCR)+, BW5147 T-lymphoma cell. High levels of expression of RPTPalpha did not fully restore either proximal or distal TCR-mediated signalling events. RPTPalpha was unable to reconstitute the phosphorylation of CD3zeta and did not increase the expression of the activation marker, CD69, on stimulation with TCR/CD3. RPTPalpha did not significantly alter the phosphorylation state or kinase activity of two CD45 substrates, p56(lck) or p59(fyn), suggesting that RPTPalpha does not have the same specificity or function as CD45 in T-cells. Further comparison of the two phosphatases indicated that immunoprecipitated RPTPalpha was approx. one-seventh to one-tenth as active as CD45 when tested against artificial substrates. This difference in activity was also observed in vitro with purified recombinant enzymes at physiological pH. Additional analysis with Src family phosphopeptides and recombinant p56(lck) as substrates indicated that CD45 was consistently more active than RPTPalpha, having both higher Vmax and lower Km values. Thus CD45 is intrinsically a much more active phosphatase than RPTPalpha, which provides one reason why RPTPalpha cannot effectively dephosphorylate p56(lck) and substitute for CD45 in T-cells. This work establishes that these two related protein tyrosine phosphatases are not interchangeable in T-cells and that this is due, at least in part, to quantitative differences in phosphatase activity.