LZAP and PPM phosphatases: reciprocal regulation and shared mechanisms altering tumor behavior

Abstract

LZAP (Cdk5rap3, C53) is a putative tumor suppressor lost in 30% of human head and neck squamous cell carcinoma. LZAP depletion enhances cell invasion, xenograft tumor growth and angiogenesis. Mechanistically, LZAP inhibits activity of NF-κB and Chk1 and Chk2 through binding and decreasing phosphorylation of critical proteins. LZAP has no known enzymatic activity, implying that its biological functions are likely mediated by protein-protein interactions. The focus of this dissertation is to better understand LZAP activity through discovery of LZAP-associated proteins. Immunoprecipitation followed by liquid chromatography¨Cmass spectrometry identified members of protein phosphatase 2C family, PPM1A and PPM1B, as LZAP binding proteins Wip1/PPM1D, a known RelA phosphatase is a member of this family. Here we show that PPM1A dephosphorylates S536 and S276 of RelA and selectively inhibits NF-κB transcriptional activity resulting in decreased expression of cytokines implicated in cancer metastases. In human prostate cancer, metastatic deposits had lower PPM1A mRNA compared to primary tumors without metastases. In xenograft models, PPM1A expression inhibited bony metastases, whereasPPM1A depletion enhanced tumor growth. Interestingly, activities of LZAP and PPM1A to inhibit RelA inhibition are mutually dependent. LZAP contains motifs predicted to bind the docking domain (D domain) of mitogen activated protein kinases (MAPKs). LZAP binds and inhibit p38 phosphorylation, alters p38 cellular localization, and inhibits basal and cytokine-stimulated p38 activity. LZAP binds the p38 phosphatase, Wip1, and facilitates Wip1 binding and dephosphorylation of p38. These data suggest that LZAP inhibition of p38 phosphorylation and activity depends, at least partially, on Wip1. Regulation of LZAP activity is not well understood; however, we found that LZAP protein is modified by phosphorylation and ubiquitination, forms dimers/oligomers, and may be a target of PPM family members. These findings provide insight into LZAP activity and may lead to mechanisms to restore LZAP activity. Together, this work suggests a common mechanism of LZAP activity to regulate important cancer-related proteins, namely interaction with and regulation of PPM phosphatase family members.