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The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the pRB tumor suppressor protein. Cyclin D1 is overexpressed in 20 -30% of human breast tumors and is induced both by oncogenes including those for Ras, Neu, and Src, and by the ␤-catenin/lymphoid enhancer factor (LEF)/T cell factor (TCF) pathway. The ankyrin repeat containing serine-threonine protein kinase, integrinlinked kinase (ILK), binds to the cytoplasmic domain of ␤ 1 and ␤ 3 integrin subunits and promotes anchorageindependent growth. We show here that ILK overexpression elevates cyclin D1 protein levels and directly induces the cyclin D1 gene in mammary epithelial cells. ILK activation of the cyclin D1 promoter was abolished by point mutation of a cAMP-responsive element-binding protein (CREB)/ATF-2 binding site at nucleotide ؊54 in the cyclin D1 promoter, and by overexpression of either glycogen synthase kinase-3␤ (GSK-3␤) or dominant negative mutants of CREB or ATF-2. Inhibition of the PI 3-kinase and AKT/protein kinase B, but not of the p38, ERK, or JNK signaling pathways, reduced ILK induction of cyclin D1 expression. ILK induced CREB transactivation and CREB binding to the cyclin D1 promoter CRE. Wnt-1 overexpression in mammary epithelial cells induced cyclin D1 mRNA and targeted overexpression of Wnt-1 in the mammary gland of transgenic mice increased both ILK activity and cyclin D1 levels. We conclude that the cyclin D1 gene is regulated by the Wnt-1 and ILK signaling pathways and that ILK induction of cyclin D1 involves the CREB signaling pathway in mammary epithelial cells.The cyclin D1 gene encodes a regulatory subunit of a serinethreonine kinase that phosphorylates and inactivates the tumor suppressor pRB (1). The abundance of cyclin D1 was shown to be rate-limiting in cellular proliferation induced by diverse signaling pathways in fibroblasts and breast epithelial cells, including MCF7 cells (2, 3). Homozygous deletion of the cyclin D1 gene in mice results in defects in mammary gland development (4, 5) and in serum-induced cellular proliferation (6). The abundance of cyclin D1 is increased in more than 30% of human breast tumors, and overexpression of cyclin D1 under control of the MMTV 1 promoter in transgenic mice induces mammary adenocarcinoma (7). The majority of breast cancer cell lines and mammary tumors induced by transgenic overexpression of either pp60 v-src or ErbB-2 oncogenes overexpress cyclin D1, suggesting that the induction of cyclin D1 may play an important role in mammary tumorigenesis (8). The cyclin D1 gene is activated by mitogenic stimuli induced by G-protein-

Caveolin-1 was first identified as a phosphoprotein in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. Tyrosine 14 is now thought to be the principal site for recognition by c-Src kinase; however, little is known about this phosphorylation event. Here, we generated a monoclonal antibody (mAb) probe that recognizes only tyrosine 14-phosphorylated caveolin-1. Using this approach, we show that caveolin-1 (Y14) is a specific tyrosine kinase substrate that is constitutively phosphorylated in Src- and Abl-transformed cells and transiently phosphorylated in a regulated fashion during growth factor signaling. We also provide evidence that tyrosine-phosphorylated caveolin-1 is localized at the major sites of tyrosine-kinase signaling, i.e. focal adhesions. By analogy with other signaling events, we hypothesized that caveolin-1 could serve as a docking site for pTyr-binding molecules. In support of this hypothesis, we show that phosphorylation of caveolin-1 on tyrosine 14 confers binding to Grb7 (an SH2-domain containing protein) both in vitro and in vivo. Furthermore, we demonstrate that binding of Grb7 to tyrosine 14-phosphorylated caveolin-1 functionally augments anchorage-independent growth and epidermal growth factor (EGF)-stimulated cell migration. We discuss the possible implications of our findings in the context of signal transduction.

Caveolin-1 is the principal protein component of caveolae membrane domains, which are located at the cell surface in most cell types. Evidence has accumulated suggesting that caveolin-1 may function as a suppressor of cell transformation in cultured cells. The human CAV-1 gene is located at a putative tumor suppressor locus (7q31.1/D7S522) and a known fragile site (FRA7G) that is deleted in a variety of epithelial-derived tumors. Mechanistically, caveolin-1 is known to function as a negative regulator of the Ras-p42/44 MAP kinase cascade and as a transcriptional repressor of cyclin D1, possibly explaining its transformation suppressor activity in cultured cells. However, it remains unknown whether caveolin-1 functions as a tumor suppressor gene in vivo. Here, we examine the tumor suppressor function of caveolin-1 using Cav-1 (-/-) null mice as a model system. Cav-1 null mice and their wild-type counterparts were subjected to carcinogen-induced skin tumorigenesis, using 7,12-dimethylbenzanthracene (DMBA). Mice were monitored weekly for the development of tumors. We demonstrate that Cav-1 null mice are dramatically more susceptible to carcinogen-induced tumorigenesis, as they develop skin tumors at an increased rate. After 16 weeks of DMBA-treatment, Cav-1 null mice showed a 10-fold increase in tumor incidence, a 15-fold increase in tumor number per mouse (multiplicity), and a 35-fold increase in tumor area per mouse, as compared with wild-type littermate mice. Moreover, before the development of tumors, DMBA-treatment induced severe epidermal hyperplasia in Cav-1 null mice. Both the basal cell layer and the suprabasal cell layers were expanded in treated Cav-1 null mice, as evidenced by immunostaining with cell-type specific differentiation markers (keratin-10 and keratin-14). In addition, cyclin D1 and phospho-ERK1/2 levels were up-regulated during epidermal hyperplasia, suggesting a possible mechanism for the increased susceptibility of Cav-1 null mice to tumorigenesis. However, the skin of untreated Cav-1 null mice appeared normal, without any evidence of epidermal hyperplasia, despite the fact that Cav-1 null keratinocytes failed to express caveolin-1 and showed a complete ablation of caveolae formation. Thus, Cav-1 null mice require an appropriate oncogenic stimulus, such as DMBA treatment, to reveal their increased susceptibility toward epidermal hyperplasia and skin tumor formation. Our results provide the first genetic evidence that caveolin-1 indeed functions as a tumor suppressor gene in vivo.