Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis

Abstract

The protein kinase PINK1 was recently shown to phosphorylate ubiquitin (Ub) on Ser65, and phosphoUb activates the E3 ligase Parkin allosterically. Here, we show that PINK1 can phosphorylate every Ub in Ub chains. Moreover, Ser65 phosphorylation alters Ub structure, generating two conformations in solution. A crystal structure of the major conformation resembles Ub but has altered surface properties. NMR reveals a second phosphoUb conformation in which β5-strand slippage retracts the C-terminal tail by two residues into the Ub core. We further show that phosphoUb has no effect on E1-mediated E2 charging but can affect discharging of E2 enzymes to form polyUb chains. Notably, UBE2R1- (CDC34), UBE2N/UBE2V1- (UBC13/UEV1A), TRAF6- and HOIP-mediated chain assembly is inhibited by phosphoUb. While Lys63-linked poly-phosphoUb is recognized by the TAB2 NZF Ub binding domain (UBD), 10 out of 12 deubiquitinases (DUBs), including USP8, USP15 and USP30, are impaired in hydrolyzing phosphoUb chains. Hence, Ub phosphorylation has repercussions for ubiquitination and deubiquitination cascades beyond Parkin activation and may provide an independent layer of regulation in the Ub system.

Structure of phosphorylated UBL domain and insights into PINK1-orchestrated parkin activation

“…4A, black and red). To complement these studies, we calculated hydrodynamic properties of autoinhibited parkin, using available 3D structures (15,21,34). Results from this approach (3.86 S, R G = 27Å) matched nearly perfectly with the experimental data for parkin and pParkin (Fig.…”

“…S2C) (21). In pUb the structural hydrogen bond from pSer65 to the backbone amide of Q62 is preserved, maintaining the orientation of the surrounding backbone residues.…”

“…Threedimensional structures of parkin show UBL associates with the C-terminal region (R0RBR) through both ionic and hydrophobic interactions, blocking the proposed E2 recognition site (14,15). PINK1 stimulates parkin activity through phosphorylation of both Ub and parkin's UBL domain at an equivalent serine 65 position in sequence and structure (16)(17)(18)(19)(20)(21). Whereas each phosphorylation event can increase parkin activity independently, maximal activity is obtained when both parkin and Ub are phosphorylated (18,19).…”

Proteomic techniques to probe the ubiquitin landscape

“…A number of other large-scale studies extended the list of modification to phosphorylations on serines, threonine and tyrosines [120][121][122], acetylation on all internal lysines [123] and the modification of lysine 11 by SUMO [124], another ubiquitin-like modification. Serine 65 has been shown to be phosphorylated by the PINK1 kinase [125], and is induced after mitochondrial depolarization [126]. The inclusion of phosphorylated ubiquitin into the ubiquitin chain alters the structure, generating a different signal [125].…”

“…Serine 65 has been shown to be phosphorylated by the PINK1 kinase [125], and is induced after mitochondrial depolarization [126]. The inclusion of phosphorylated ubiquitin into the ubiquitin chain alters the structure, generating a different signal [125]. This can change the polymerization of the chain as well as the overall amount of ubiquitination in the cell, while the overall degradation rates decrease [127].…”