Mechanistic Insights into the Auto-Regulation of PTEN and Its Related HECT E3 Ligases
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PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a lipid phosphatase that dephosphorylates PIP3 to PIP2, and negatively regulates the PI3K/AKT signaling pathway. PTEN is an essential tumor suppressor gene frequently deactivated in cancer cells. Previous work in our lab and by others has shed light on the regulation of PTEN by a cluster of phosphorylation sites within the C-terminal regulatory tail at residues Ser380, Thr382, Thr383 and Ser385. Tetra-phosphorylation of the C-terminal tail binds to the C2 domain and leads to a closed conformation which inhibits PTEN’s lipid phosphatase activity and membrane association, but increases its stability. We continued to investigate the molecular mechanisms of this regulatory mechanism using native chemical ligation. Systematic evaluation of the PTEN C-tail phospho-cluster showed that the conformational closure and autoinhibition was influenced by the aggregate effect of multiple phospho-sites rather than dominated by a single phosphorylation site. The photo-crosslinking results suggested that the phosphorylated C-tail not only interacts with the C2 domain but also the N-terminal phosphatase domain. The ubiquitination assays analyzing PTEN ubiquitination by WWP2 showed that PTEN C-tail phosphorylation could inhibit its ubiquitination by the HECT E3 ligases, presumably by disrupting the protein-protein interaction between PTEN and WWP2. In the course of analyzing PTEN ubiquitination by WWP2, we noticed that the monomeric WWP2 is autoinhibited. WWP2 is a member of the NEDD4 HECT E3 ligase family, and the autoinhibition mechanisms of this family have been reported as involving intramolecular interactions between C2 or WW domains with the corresponding catalytic HECT domains. However, we found unexpectedly that a peptide linker tethering WW domains is the key regulatory element of WWP2 catalytic activity. Biochemical, structural, and cellular analyses have revealed that the linker can lock the HECT domain in an inactive conformation and block the proposed allosteric ubiquitin binding site. Such linker mediated autoinhibition can be relieved by linker post-translational modifications, but complete removal of the linker can induce hyperactive autoubiquitination and E3 self-destruction. We further showed that this regulatory mechanism is not limited to WWP2, but also applies to the related HECT family members WWP1, ITCH, and NEDD4-1.