Abstract 2407: Serine and threonine phosphorylation marks proteins for degradation by ClpXP
Authors: Feng Yue , Jitkova Yulia , Schimmer Aaron D. 
Published: July 2021

Abstract
The mitochondrial serine protease ClpXP regulates the integrity of the respiratory chain by degrading damaged and/or misfolded proteins. This protease is over-expressed in multiple malignancies and inhibiting or hyperactivating it kills malignant cells in vitro and in vivo. In Bacillus subtilis, the bacterial ClpXP homologue recognizes proteins tagged with phosphor-arginine for degradation. Yet, it is unknown how the mitochondrial ClpXP recognizes proteins for degradation.
To determine how phosphorylated amino acids influence ClpXP-mediated protein degradation, we incubated recombinant ClpXP with its unnatural substrate FITC-casein and increasing concentrations of phospho-serine (pSer), phospho-threonine (pThr), phospho-arginine (pArg), or phosphor-tryrosine (pTry) in a cell-free assay and measured release of fluorogenic FITC. In a dose-dependent manner, pSer and pThr free amino acids inhibited casein cleavage by ClpXP while pTyr, pArg, and the dephosphorylated amino acids had no effect on ClpXP activity. Likewise, ApSA and ApTA peptides inhibited ClpXP enzyme activity, while the non-phosphorylated version (ASA and ATA) and no effect.
Next, we tested whether the phosphorylation state of full length proteins would influence their degradation by ClpXP. Using gel-based cell-free assays, the phosphorylation enriched α-casein and β-casein were degraded by recombinant ClpXP. In contrast, the κ-casein with low phosphorylation level and dephosphorylated α-casein were not cleaved by ClpXP. In addition, we treated α-casein with lambda-phosphatases to dephosphorylate the protein and confirmed the release of free phosphate by the Malachite green phosphate assay. Phosphatase treated α-casein was resistant to ClpXP cleavage.
As ClpXP is an AAA ATPase, we asked if pSer and pThr acted on the ATPase of the enzyme. pSer and pThr did not inhibit the ATPase activity of ClpX, as measured by the malachite green ATPase assay. We also measured the effect of pSer and pThr on the peptidase activity of ClpP alone without its regulatory subunit ClpX. Neither pSer or pThr inhibited ClpP peptidase activity.
We investigated if pSer and pThr could bind to ClpX. Using thermal shift binding assays, we demonstrated that pSer and pThr but not pTyr and pArg bind ClpX and none of the phosphorylated amino acids bind ClpP.
Finally, we characterized the total and phopho- mitochondrial proteome of T-REX HEK293 wild type and ClpP knockout cells by mass spectrometry. We identified 402 unique mitochondrial proteins and 38 phosphorylated mitochondrial proteins in wild type cells. 20 phosphorylated mitochondrial proteins increased in the ClpP knockout cells compared to wild type with a cut-off of 2 fold.
In summary, we discovered that ClpX binds pSer and pThr and phosphorylation of these amino acids mark proteins for degradation by the ClpXP mitochondrial protease. This work highlights a new strategy to develop inhibitors of ClpXP for the treatment of cancer.
Citation Format: Yue Feng, Yulia Jitkova, Aaron D. Schimmer. Serine and threonine phosphorylation marks proteins for degradation by ClpXP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2407.