Browsing Upstate Medical University by Subject "PHOSPHORYLATION"
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PHOSPHORYLATION AND UBIQUITINATION REGULATE PROTEIN PHOSPHATASE 5 ACTIVITY AND ITS PROSURVIVAL ROLE IN KIDNEY CANCERProtein Phosphatase 5 (PP5) is a serine/threonine phosphatase known to regulate many essential cellular functions including steroid hormone signaling, stress response, proliferation, apoptosis, and DNA repair. PP5 is a knownco-chaperone of the molecular chaperone heat shock protein 90 (Hsp90), and its regulation of Hsp90aidswiththe proper activation of Hsp90 clients and withsteroid hormone signaling.Hsp90 is also one of the strongest activators of PP5, as it releases the auto-inhibition of PP5 by interacting with the N-terminal tetratricopeptide repeat (TPR) domain of PP5. Our lab has recently shown that PP5 is phosphorylated at T362, and that this phosphorylation acts as an “on switch” resultingin the hyperactivation of PP5. Misregulation of this key phosphatase has been shown to aid in the tumor progression of ER-dependent and independent breast cancer. Elevated PP5 levels have also been linked to colorectalcancer, hepatocellular carcinoma (HCC), lymphoma, and prostate cancer. The work presented here reveals the pro-survival role that PP5 plays in kidney cancer. Clear cell renal cell carcinomas (ccRCC) are most often driven by mutations in the von Hippel-Lindau tumor suppressor (VHL). The data in this thesis shows that VHL binds and multi mono-ubiquitinates PP5 at two lysine residues K185 and K199. This post-translational modification negatively regulates PP5 likean “off switch” and ultimately leads to its degradation bythe proteasome. Mutations in the VHLgene that result in inactive mutants or a lack of VHL protein expression lead to ccRCC tumors. The data in this thesis shows that these VHL-nulltumors become dependent on elevated levels of PP5, and that both PP5 knockdown and inhibition lead to cancer cell death. The data further shows that the decrease in PP5 activity in VHL-null cells results in the induction of the extrinsic apoptotic pathway with a dramatic increase in the cleavage of PARP and caspases 3, 7, and 8.
TIMP-2 PHOSPHORYLATION BY EXTRACELLULAR c-SRC REGULATES proMMP-2 ACTIVATIONMatrix metalloproteinases (MMPs) are secreted zinc-dependent endopeptidases that are involved in many extracellular biological processes due to their matrix-degrading function. The majority of theseenzymes are released intothe extracellular space in their inactive form and require activation. The tissue inhibitors of metalloproteinases (TIMPs) are also secreted proteins and mainly function to inhibit all members of the MMP family. Interestingly, TIMP-2 also participates in the activationprocessof proMMP-2. Although the interaction between TIMP-2 and proMMP-2 has been known for decades, the molecular signal that triggersthis association has only recently beendetermined. Studies in our lab haveshown that TIMP-2 is tyrosine phosphorylatedby the c-Srctyrosinekinase. Also, phosphorylation of TIMP-2 Tyr90is essential for its interaction with proMMP-2in vivo. Our hypothesis is that c-Src-mediated TIMP-2 phosphorylation happens outside the cell. Here, wedemonstratethat TIMP-2 and c-Src are secreted through different secretory pathways and that TIMP-2 phosphorylation takes place in the extracellular space. Our workalso showsthatextracellularc-Srcisactive, reinforcing the fact that phosphorylation can happen extracellularly. We also hypothesize that extracellular c-Src plays a critical role in facilitating TIMP-2:proMMP-2 interaction. We first confirmed thatTIMP-2 and proMMP-2 endogenously interact only in cells containing endogenous c-Src. This interaction,as well as TIMP-2 phosphorylation,was blocked by treating cells with acustom-made anti-c-Src polyclonal antibody (pAb)that targets amino acids 84-110. We also showthat ananti-c-Src antibody that targets the first 79 amino acids does not inhibit TIMP-2 phosphorylation and interaction with proMMP-2. Therefore, since TIMP-2:proMMP-2 complex formation promotes proMMP-2 activation, we hypothesize that c-Src is an essential player in this process. Our data showsthatthe non-phosphorylatable TIMP-2Tyr90mutant does not promote proMMP-2 activation. Furthermore, pretreatment with the anti-c-Src pAbblockedTIMP-2-mediated proMMP-2 activation, whereasthe anti-c-Src mAb6 did not affect proMMP-2 activation. Overall, these findings provide further evidence that secreted c-Src-mediated TIMP-2 phosphorylation occurs in the extracellular space, where thesecretedkinase is also active. Moreover, c-Src is essential for TIMP-2:proMMP-2 complex formation as well as proMMP-2 activation.