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USP14 is phosphorylated on S432. 29 / 29
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"Finally, USP14 S432 is dramatically more phosphorylated in PTEN knockout mouse embryonic fibroblasts (MEFs), which carry high levels of Akt activity, than that of WT MEFs as determined by western blotting using the phospho-USP14(S432) antibody and phos-tag electrophoresis ( xref ), and the phosphorylation of USP14 S432 was blocked by Akt inhibitors ( xref )."
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"(E) S432 is the major phosphorylation site in USP14."
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"(F) In vivo detection of endogenous USP14 Ser432 phosphorylation by anti-p-Ser432-specific antibody."
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"We found that the treatment of IGF-1 or EGF resulted in phosphorylation of USP14 S432, which was blocked in cells pretreated with MK2206 ( xref )."
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"Thus, S432 phosphorylated and unphosphorylated USP14 might be distributed differently in the cells. We next determined whether phospho-mimetic mutant of USP14 could be further activated by interacting with proteasome. Interestingly, we found that the Ub-AMC hydrolytic activity of S432E mutant could be further activated when incubated with proteasome in vitro (Figure 3H). Taken together, these results suggest that S432 phosphorylation and interaction with proteasome may be two different regulatory mechanisms for USP14."
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"Akt-mediated phosphorylation of USP14 at Ser432 activates its deubiquitinating activity in vitro and in cells ( xref )."
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"Structural basis of ubiquitin-specific protease-14 (USP14) activation by phosphorylation of Ser432."
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"(G, H) Phosphorylation of endogenous USP14 S432 upon stimulation with insulin-like growth factor (IGF-1) or epidermal growth factor (EGF)."
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"Xu et al. reported that serine/threonine kinase could phosphorylate USP14 at Ser432, thus activating its deubiquitinating activity by transforming its catalytic site from the inactive conformation to the active form. xref Whether the activity of USP14 is regulated by serine/threonine kinase in ESCC is still questionable."
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"Expression of Myr-Akt also led to S432 phosphorylation of endogenous USP14 ( xref )."
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"The reaction products were resolved by SDS-PAGE, and USP14 phosphorylation was detected using an antibody that specifically recognizes Ser432 phosphorylation of USP14 or determined by differential migration on phos-tag gels."
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"When S432 is phosphorylated, the negatively charged phosphate group may induce a repulsive force, thereby relieving inhibition of the catalytic activity of USP14. (D) USP14 domain organization and sequence alignment of the Akt phosphorylation site within USP14 orthologs from different species. Two BLs (BL1 and BL2) covering the USP14 active site are shown. The Akt phosphorylation site in USP14 from different species as predicted by Scansite. (E) S432 is the major phosphorylation site in USP14."
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"They further show that USP14 is phosphorylated constitutively at Ser432 in PTEN null cells."
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"Interestingly, human USP14 contains a total of seven phosphorylation sites of which the Akt-mediated USP14 phosphorylation at Ser432 activates its DUB activity and facilitates cleavage preferentially towards Lys48- and Lys63-linked chains rather than linear ubiquitin chains ( xref )."
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"Moreover, treatment with PI3K inhibitors, either Wortmannin or GDC0941, but not ERK1/2 inhibitor U0126, also significantly decreased the phosphorylation levels of USP14 S432 (Figure 2—figure supplement 1D,E)."
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"It has been reported that AKT mediates phosphorylation of USP14 on Ser432 ( xref )."
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"We report that Akt-mediated phosphorylation of USP14 at Ser432, which normally blocks its catalytic site in the inactive conformation, activates its deubiquitinating activity in vitro and in cells."
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"Among them, the Akt-mediated USP14 phosphorylation at Ser432 activates its deubiquitinating activity and facilitates cleavage towards K48- and K63-linked chains rather than linear ubiquitin chains ( xref ) ( xref )."
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"We identified four phosphorylation sites on USP14 when it was expressed alone: Ser143, Ser230, Thr235, and Ser432 (Figure 1—figure supplement 1C,D). Notably, the phosphorylation levels of two of the four sites, Ser143 and Ser432, were increased considerably in cells expressing activated Akt (Figure 1E)."
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"To further verify the phosphorylation of USP14 S432 by Akt, we developed a phospho-Ser432-specific antibody. Phosphorylation of S432 can be detected after incubation of WT, but not S432A mutant USP14, with recombinant activated Akt in a kinase reaction (Figure 2E)."
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"(I) Phosphorylation of endogenous USP14 S432 in Pten knockout cells with high activity of Akt."
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"Thus, S432 phosphorylated and unphosphorylated USP14 might be distributed differently in the cells."
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"Finally, USP14 S432 is dramatically more phosphorylated in PTEN knockout mouse embryonic fibroblasts (MEFs), which carry high levels of Akt activity, than that of WT MEFs as determined by western blotting using the phospho-USP14(S432) antibody and phos-tag electrophoresis (Figure 2I), and the phosphorylation of USP14 S432 was blocked by Akt inhibitors (Figure 2—figure supplement 1F)."
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"Expression of Myr-Akt also led to S432 phosphorylation of endogenous USP14 (Figure 2F)."
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"We found that the treatment of IGF-1 or EGF resulted in phosphorylation of USP14 S432, which was blocked in cells pretreated with MK2206 (Figure 2G,H)."
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"Treatment with either MK2206 or AZD5363, two structurally unrelated Akt inhibitors, led to decrease of USP14 S432 phosphorylation levels (Figure 2—figure supplement 1B,C)."
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"Since Ser432 residue is located very close to a highly negatively charged patch (Figure 1C), we reasoned that when Ser432 residue was phosphorylated, the negatively charged phosphate group might induce a repulsive force, thereby inducing rearrangement of the BL2 loop and removing the inhibitory effect of this loop on the activity of USP14."
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"We identified four phosphorylation sites on USP14 when it was expressed alone: Ser143, Ser230, Thr235, and Ser432 (Figure 1—figure supplement 1C,D)."
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"1) The data presented in the paper provide convincing evidence that the phosphorylation of USP14 at Ser432, and perhaps also Ser143, activates USP14 activity in vitro and in overexpression experiments in cells."
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