SUMMARY Protein interactions form a network whose structure drives cellular function and whose organization informs biological inquiry. Using high-throughput affinity-purification mass spectrometry, we identify interacting partners for 2,594 human proteins in HEK293T cells. The resulting network (BioPlex) contains 23,744 interactions among 7,668 proteins with 86% previously undocumented. BioPlex accurately depicts known complexes, attaining 80-100% coverage for most CORUM complexes. The network readily subdivides into communities that correspond to complexes or clusters of functionally related proteins. More generally, network architecture reflects cellular localization, biological process, and molecular function, enabling functional characterization of thousands of proteins. Network structure also reveals associations among thousands of protein domains, suggesting a basis for examining structurally-related proteins. Finally, BioPlex, in combination with other approaches can be used to reveal interactions of biological or clinical significance. For example, mutations in the membrane protein VAPB implicated in familial Amyotrophic Lateral Sclerosis perturb a defined community of interactors.

Phosphorylation is often used to promote protein ubiquitylation, yet we rarely understand quantitatively how ligase activation and ubiquitin (UB) chain assembly are integrated with phospho-regulation. Here we employ quantitative proteomics and live-cell imaging to dissect individual steps in the PINK1 kinase-PARKIN UB ligase mitochondrial control pathway disrupted in Parkinson’s Disease. PINK1 plays a dual role by phosphorylating PARKIN on its UB-like domain and poly-UB chains on mitochondria. PARKIN activation by PINK1 produces canonical and non-canonical UB chains on mitochondria, and PARKIN-dependent chain assembly is required for accumulation of poly-phospho-UB (poly-p-UB) on mitochondria. In vitro, PINK1 directly activates PARKIN’s ability to assemble canonical and non-canonical UB chains, and promotes association of PARKIN with both p-UB and poly-p-UB. Our data reveal a feed-forward mechanism that explains how PINK1 phosphorylation of both PARKIN and poly-UB chains synthesized by PARKIN drives a program of PARKIN recruitment and mitochondrial ubiquitylation in response to mitochondrial damage.

Damaged mitochondria are detrimental to cellular homeostasis. One mechanism for removal of damaged mitochondria involves the PINK1-PARKIN pathway, which poly-ubiquitylates damaged mitochondria to promote mitophagy. We report that assembly of ubiquitin chains on mitochondria triggers the recruitment of autophagy adaptors concomitantly with activation of the TBK1 protein kinase, which physically associates with OPTN, NDP52, and SQSTM1. Full TBK1 activation in HeLa cells requires OPTN and NDP52, and OPTN ubiquitin chain binding. In addition to the known role of S177 phosphorylation in OPTN on ATG8 recruitment, TBK1-dependent phosphorylation on S473 and S513 promotes ubiquitin chain binding in vitro as well as TBK1 activation, OPTN mitochondrial retention, and efficient mitophagy in vivo. These data reveal a self-reinforcing positive feedback mechanism that coordinates TBK1-dependent autophagy adaptor phosphorylation with the assembly of ubiquitin chains on mitochondria to facilitate efficient mitophagy. Analogous mechanisms may facilitate adaptor-protein mediated delivery of other types of cargo to autophagosomes.

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment. Here we report the results of a moderate-scale sequencing study aimed at identifying new genes contributing to predisposition for ALS. We performed whole exome sequencing of 2,874 ALS patients and compared them to 6,405 controls. Several known ALS genes were found to be associated, and the non-canonical IκB kinase family TANK-Binding Kinase 1 (TBK1) was identified as an ALS gene. TBK1 is known to bind to and phosphorylate a number of proteins involved in innate immunity and autophagy, including optineurin (OPTN) and p62 (SQSTM1/sequestosome), both of which have also been implicated in ALS. These observations reveal a key role of the autophagic pathway in ALS and suggest specific targets for therapeutic intervention.

LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinson's disease patients, but still little is understood about how it is regulated or functions. In the present study we have demonstrated that 14-3-3 protein isoforms interact with LRRK2. Consistent with this, endogenous LRRK2 isolated from Swiss 3T3 cells or various mouse tissues is associated with endogenous 14-3-3 isoforms. We have established that 14-3-3 binding is mediated by phosphorylation of LRRK2 at two conserved residues (Ser910 and Ser935) located before the leucine-rich repeat domain. Our results suggests that mutation of Ser910 and/or Ser935 to disrupt 14-3-3 binding does not affect intrinsic protein kinase activity, but induces LRRK2 to accumulate within discrete cytoplasmic pools, perhaps resembling inclusion bodies. To investigate links between 14-3-3 binding and Parkinson's disease, we studied how 41 reported mutations of LRRK2 affected 14-3-3 binding and cellular localization. Strikingly, we found that five of the six most common pathogenic mutations (R1441C, R1441G, R1441H, Y1699C and I2020T) display markedly reduced phosphorylation of Ser910/Ser935 thereby disrupting interaction with 14-3-3. We have also demonstrated that Ser910/Ser935 phosphorylation and 14-3-3 binding to endogenous LRRK2 is significantly reduced in tissues of homozygous LRRK2(R1441C) knock-in mice. Consistent with 14-3-3 regulating localization, all of the common pathogenic mutations displaying reduced 14-3-3-binding accumulated within inclusion bodies. We also found that three of the 41 LRRK2 mutations analysed displayed elevated protein kinase activity (R1728H, ~2-fold; G2019S, ~3-fold; and T2031S, ~4-fold). These results provide the first evidence suggesting that 14-3-3 regulates LRRK2 and that disruption of the interaction of LRRK2 with 14-3-3 may be linked to Parkinson's disease.

Polyubiquitin (pUb) chains formed between the C terminus of ubiquitin and lysine 63 (K63) or methionine 1 (M1) of another ubiquitin have been implicated in the activation of the canonical IκB kinase (IKK) complex. Here, we demonstrate that nearly all of the M1-pUb chains formed in response to interleukin-1, or the TollLike Receptors 1/2 agonist Pam 3 CSK 4 , are covalently attached to K63-pUb chains either directly as K63-pUb/M1-pUb hybrids or indirectly by attachment to the same protein. Interleukin-1 receptor (IL-1R)-associated kinase (IRAK) 1 is modified first by K63-pUb chains to which M1-pUb linkages are added subsequently, and myeloid differentiation primary response gene 88 (MyD88) and IRAK4 are also modified by both K63-pUb and M1-pUb chains. We show that the heme-oxidized IRP2 ubiquitin ligase 1 interacting protein (HOIP) component of the linear ubiquitin assembly complex catalyzes the formation of M1-pUb chains in response to interleukin-1, that the formation of K63-pUb chains is a prerequisite for the formation of M1-pUb chains, and that HOIP interacts with K63-pUb but not M1-pUb linkages. These findings identify K63-Ub oligomers as a major substrate of HOIP in cells where the MyD88-dependent signaling network is activated. The TGFbeta-activated kinase 1 (TAK1)-binding protein (TAB) 2 and TAB3 components of the TAK1 complex and the NFκB Essential Modifier (NEMO) component of the canonical IKK complex bind to K63-pUb chains and M1-pUb chains, respectively. The formation of K63/M1-pUb hybrids may therefore provide an elegant mechanism for colocalizing both complexes to the same pUb chain, facilitating the TAK1-catalyzed activation of IKKα and IKKβ. Our study may help to resolve the debate about the relative importance of K63-pUb and M1-pUb chains in activating the canonical IKK complex.LUBAC | TNF Receptor-Associated Factor 6 | Ubc13 | NF-κB

Mitochondria produce energy in the form of ATP via oxidative phosphorylation. As defects in oxidative phosphorylation can generate harmful reactive oxygen species, it is important that damaged mitochondria are efficiently removed via a selective form of autophagy known as mitophagy. Owing to a combination of cell biological, structural and proteomic approaches, we are beginning to understand the mechanisms by which ubiquitin-dependent signals mark damaged mitochondria for mitophagy. This Review discusses the biochemical steps and regulatory mechanisms that promote the conjugation of ubiquitin to damaged mitochondria via the PTEN-induced putative kinase 1 (PINK1) and the E3 ubiquitin-protein ligase parkin and how ubiquitin chains promote autophagosomal capture. Recently discovered roles for parkin and PINK1 in the suppression of mitochondrial antigen presentation provide alternative models for how this pathway promotes the survival of neurons. A deeper understanding of these processes has major implications for neurodegenerative diseases, including Parkinson disease, where defects in mitophagy and other forms of selective autophagy are prominent.

The PTEN-induced putative kinase protein 1 (PINK1) and ubiquitin (UB) ligase PARKIN direct damaged mitochondria for mitophagy. PINK1 promotes PARKIN recruitment to the mitochondrial outer membrane (MOM) for ubiquitylation of MOM proteins with canonical and noncanonical UB chains. PINK1 phosphorylates both Ser65 (S65) in the UB-like domain of PARKIN and the conserved Ser in UB itself, but the temporal sequence and relative importance of these events during PARKIN activation and mitochondria quality control remain poorly understood. Using "UB S65A-replacement," we find that PARKIN phosphorylation and activation, and ubiquitylation of Lys residues on a cohort of MOM proteins, occur similarly irrespective of the ability of the UB-replacement to be phosphorylated on S65. In contrast, polyubiquitin (poly-UB) chain synthesis, PARKIN retention on the MOM, and mitophagy are reduced in UB S65A -replacement cells. Analogous experiments examining roles of individual UB chain linkage types revealed the importance of K6 and K63 chain linkages in mitophagy, but phosphorylation of K63 chains by PINK1 did not enhance binding to candidate mitophagy receptors optineurin (OPTN), sequestosome-1 (p62), and nuclear dot protein 52 (NDP52) in vitro. Parallel reaction monitoring proteomics of total mitochondria revealed the absence of p-S65-UB when PARKIN cannot build UB chains, and <0.16% of the monomeric UB pool underwent S65 phosphorylation upon mitochondrial damage. Combining p-S65-UB and p-S65-PARKIN in vitro showed accelerated transfer of nonphosphorylated UB to PARKIN itself, its substrate mitochondrial Rho GTPase (MIRO), and UB. Our data further define a feed-forward mitochondrial ubiquitylation pathway involving PARKIN activation upon phosphorylation, UB chain synthesis on the MOM, UB chain phosphorylation, and further PARKIN recruitment and enzymatic amplification via binding to phosphorylated UB chains.he RING-Between-RING ubiquitin (UB) ligase PARKIN and the PTEN-induced putative kinase protein 1 (PINK1), both of which are mutated in Parkinson's disease, promote ubiquitylation of numerous outer membrane proteins on damaged mitochondria, which facilitates mitophagy (1). When mitochondria are healthy, cytoplasmic PARKIN is thought to be in an unphosphorylated and auto-inhibited state (2-6). Upon mitochondrial damage, PINK1 is stabilized on the mitochondrial outer membrane (MOM) and promotes phosphorylation of both Ser65 (S65) on PARKIN's UB-like (UBL) domain and the conserved S65 residue in UB itself, which is 62% similar to PARKIN's UBL (1,(7)(8)(9)(10)(11)(12)(13)(14). PARKIN is also retained on the mitochondrial surface and ubiquitylates numerous MOM proteins through the formation of both canonical and noncanonical UB chains. The order of events, as well as the precise roles of UB and PARKIN phosphorylation, is poorly defined, and multiple models have been proposed. All models agree that PARKIN activation reverses auto-inhibition but differ in the mechanism and role of p-S65-UB (8,(11)(12)(13)(14)(15)). In one model, binding...