Abstract 3063: Novel putative tumor suppressor LZAP binds and regulates Wip1

Abstract

Abstract Our group has shown that LZAP is lost in ∼30% of head and neck squamous cell carcinomas and that inhibition of LZAP expression is associated cellular transformation and xenograft tumor growth. LZAP increases invasion and inhibits cellular proliferation and clonogenic growth mediated correlating with inhibition of NF-kB and activation of p53. Other researchers have found that LZAP promotes cell death in response to cytotoxic therapy at least partially dependent on activation of Cdk1 through Chk1/2 inhibition. We have also found that LZAP binds and inhibits p38 and this inhibition does not depend on upstream regulators MKK3 and MKK6. The activity of MAPKs reflects a balance between the upstream activating kinases and inactivating protein phosphatases. Since LZAP inhibition of p38 is not through upstream kinases, we determined if phosphatase activity targeting p38 was regulated by LZAP. Wip1 is a major phosphatase regulating p38 phosphorylation and activation. Wip1 is a member of the PP2C family of serine/threonine phosphatases and was identified in a screen to detect p53 responsive genes. We noted that LZAP and Wip1 had many binding partners/targets including RelA, p38, and Chk1/Chk2 in common. Mechanisms of LZAP inhibition of RelA, p38 and Chk1/Chk2 are not well described, but expression of LZAP is associated with decreased phosphorylation of all three target proteins. To determine if LZAP could regulate Wip1 activity, we first evaluated LZAP and Wip1 interaction through immuno- or affinity precipitation. LZAP and Wip1 co-precipitated after expression in mammalian cells and bacterially expressed LZAP pulled down Wip1 generated through in vitro transcription and translation. After transient expression in mammalian cells, Wip1 and LZAP co-localized by indirect immunofluorescence staining. When expressed singly, Flag-tagged Wip1 localized to the nucleus and LZAP was excluded from the nucleolus, but localized to both cytoplasm and nucleus. When Wip1 and LZAP were co-expressed LZAP localization was altered so that nucleolar LZAP was observed. These findings indicate that co-expression of LZAP and Wip1 results in alternation of LZAP subcellular localization with resultant co-localization of these two proteins to nucleus. Functionally, co-expression of LZAP with Wip1 increased Wip1 content within p38 immunocomplexes which was associated with decreased p38 phosphorylation. Together, our findings indicate LZAP binds and co-localizes with Wip1 and increases activity of Wip1 towards p38. Wip1 may play a common mediator for LZAP activities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3063.