Browsing by Author "Heering, Johanna"

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Open Access
Identification of novel protein binding partners for the tumor suppressor DLC1
(2009) Heering, Johanna; Pfizenmaier, Klaus (Prof. Dr.)
Cancer, one of the major chronic health problems worldwide, is a genetic disease, which requires the cooperation of gain-of-function and/or loss-of-function mutations to either oncogenes or tumor suppressor genes, respectively. In the last decade, the Deleted in Liver Cancer (DLC) 1 gene has emerged as a novel tumor suppressor downregulated in a variety of cancer types including breast, liver, prostate and lung. DLC1 is a multidomain protein consisting of an N-terminal sterile alpha motif (SAM), a C-terminal START and an internal Rho GTPase activating (GAP) domain. Due to GAP-dependent and -independent mechanisms that are still poorly understood DLC1 is able to inhibit proliferation, migration and invasion of tumor cells. The goal of this thesis was the identification of novel DLC1 protein binding partners, in order to gain deeper insight into DLC1 regulation and molecular function. Therefore, a yeast-two-hybrid screen based on the Gal4-system was performed, using the DLC1 SAM domain as a bait and a cDNA library derived from human breast tissue as a prey. One of the 16 putative binding candidates identified was the phosphatase and tensin homolog deleted on chromosome ten (PTEN), which is also a tumor suppressor frequently deleted or mutated in sporadic tumors of the breast, prostate, endometrium and brain. PTEN consists of an enlarged catalytic site that acts as a dual-specificity phosphatase for proteins and lipids. By dephosphorylation of its major lipid substrate phosphatidylinositol (3,4,5)-trisphosphate (PIP3) the PI3K signaling pathway is downregulated and cell proliferation and survival mediated by Akt/PKB activation are inhibited. Cell spreading and cell motility are also suppressed by PTEN activity. Pulldown assays and coimmunoprecipitation of DLC1 and PTEN confirmed association of the proteins in mammalian cells. The interaction of both proteins was stimulated upon PTEN activation by PDGF treatment, correlating with their colocalization at the plasma membrane. In overexpression experiments synergistic effects of the two proteins with regard to downstream signaling could not be observed. However, simultaneous loss of DLC1 and PTEN in the non-invasive MCF7 breast carcinoma cell line using RNA interference enhanced migration in an additive manner in wounding as well as in chemotactic transwell assays compared to singly depleted cells. These results suggest that the spatio-temporally restricted formation of a DLC1/PTEN complex, simultaneously inhibiting its individual downstream targets, guarantees the synchronization of cell migration processes. Thus, loss of both proteins is proposed to facilitate malignant transformation by increasing the metastatic potential of tumor cells. Another putative DLC1 binding partner identified in the yeast-two-hybrid screen was liprin beta2 that belongs to a protein familiy comprising four alpha- and two beta-type family members, which all contain a C-terminal highly conserved liprin homology (LH) domain consisting of three SAM domains. Association of full-length DLC1 with liprin beta2 as well as with the additional family members liprin alpha1 and beta1 was confirmed biochemically by coimmunoprecipitation. Liprins are multivalent proteins that form complex structures due to homo- and heterodimerization. Additionally, alpha-type liprins interact with the LAR subfamily of receptor protein-tyrosine phosphatases, which are heterophilic receptors involved in cell adhesion and cell motility. Given the function of liprins as adaptor proteins at membrane proximal sites, it is conceivable that they contribute to DLC1 localization and/or scaffolding. It is thus of particular interest to investigate the biological impact of DLC1 binding to liprin proteins in future studies.