EYA protein complex is required for Wntless retrograde trafficking from endosomes to Golgi.

Retrograde transport of WLS (Wntless) from endosomes to trans-Golgi network (TGN) is required for efficient Wnt secretion during development. However, the molecular players connecting endosomes to TGN during WLS trafficking are limited. Here, we identified a role for Eyes Absent (EYA) proteins during retrograde trafficking of WLS to TGN in human cell lines. By using worm, fly, and zebrafish models, we found that the EYA-secretory carrier-associated membrane protein 3 (SCAMP3) axis is evolved in vertebrates. EYAs form a complex and interact with retromer on early endosomes. Retromer-bound EYA complex recruits SCAMP3 to endosomes, which is necessary for the fusion of WLS-containing endosomes to TGN. Loss of EYA complex or SCAMP3 leads to defective transport of WLS to TGN and failed Wnt secretion. EYA mutations found in patients with hearing loss form a dysfunctional EYA-retromer complex that fails to activate Wnt signaling. These findings identify the EYA complex as a component of retrograde trafficking of WLS from the endosome to TGN.

Ubiquitin-assisted phase separation of dishevelled-2 promotes Wnt signalling.

Dishvelled-2 (Dvl2) is an essential component of Wnt pathway, which controls several cell fate decisions during development, such as proliferation, survival and differentiation. Dvl2 forms higher-order protein assemblies in the cell that are critical for relaying the signal from upstream Wnt ligand-frizzled receptor binding to downstream effector ß-catenin activation. However, the precise molecular nature and contribution of Dvl2 protein assemblies during Wnt signalling is unknown. Here, we show that Dvl2 forms protein condensates driven by liquid-liquid phase separation. An intrinsically disordered region (IDR) at the N-terminus is essential for Dvl2 phase separation. Importantly, we identified the HECT-E3 ligase WWP2 as an essential driver of Dvl2 phase separation in vitro and in cells. We demonstrated that ubiquitylation of Dvl2 through K63 linkage by WWP2 is required for formation of Dvl2 condensates. Phase-separated Dvl2 activates Wnt signaling by sequestering the components of destruction complex and thus relieving ß-catenin. Together, our results reveal a ubiquitylation-dependent liquid-liquid phase separation as a new process through which Dvl2 forms condensates, which is necessary for transduction of Wnt signalling. This article has an associated First Person interview with the first author of the paper.

SHP-1 dephosphorylates histone H2B to facilitate its ubiquitination during transcription.

Dynamic regulation of phosphorylation and dephosphorylation of histones is essential for eukaryotic transcription, but the enzymes engaged in histone dephosphorylation are not fully explored. Here, we show that the tyrosine phosphatase SHP-1 dephosphorylates histone H2B and plays a critical role during transition from the initiation to the elongation stage of transcription. Nuclear-localized SHP-1 is associated with the Paf1 complex at chromatin and dephosphorylates H2B at tyrosine 121. Moreover, knockout of SHP-1, or expression of a mutant mimicking constitutive phosphorylation of H2B Y121, leads to a reduction in genome-wide H2B ubiquitination, which subsequently causes defects in RNA polymerase II-dependent transcription. Mechanistically, we demonstrate that Y121 phosphorylation precludes H2B's interaction with the E2 enzyme, indicating that SHP-1-mediated dephosphorylation of this residue may be a prerequisite for efficient H2B ubiquitination. Functionally, we find that SHP-1-mediated H2B dephosphorylation contributes to maintaining basal autophagic flux in cells through the efficient transcription of autophagy and lysosomal genes. Collectively, our study reveals an important modification of histone H2B regulated by SHP-1 that has a role during eukaryotic transcription.

Ubiquitin-independent proteasomal degradation of Spindlin-1 by the E3 ligase HACE1 contributes to cell-cell adhesion.

HECT-E3 ligases play an essential role in catalyzing the transfer of ubiquitin to protein substrates. The noncatalytic roles of HECT-E3 ligases in cells are unknown. Here, we report that a HECT-E3 ligase, HACE1, functions as an adaptor independent of its E3 ligase activity. We identified Spindlin-1, a histone reader, as a new HACE1-associated protein. Interestingly, we found that HACE1 promotes Spindlin-1 degradation via the proteasome in an ubiquitination-independent manner. Functionally, we demonstrated that the loss of HACE1 results in weak cell-cell adhesion due to Spindlin-1-mediated accumulation of GDNF, a negative regulator of cell adhesion. Together, our data suggest that HACE1 acts as a molecular adaptor and plays an important noncatalytic role in presenting selected substrates directly to the proteasome for degradation.

PPM1G forms a PPP-type phosphatase holoenzyme with B56δ that maintains adherens junction integrity.

Serine/threonine phosphatases achieve substrate diversity by forming distinct holoenzyme complexes in cells. Although the PPP family of serine/threonine phosphatase family members such as PP1 and PP2A are well known to assemble and function as holoenzymes, none of the PPM family members were so far shown to act as holoenzymes. Here, we provide evidence that PPM1G, a member of PPM family of serine/threonine phosphatases, forms a distinct holoenzyme complex with the PP2A regulatory subunit B56δ. B56δ promotes the re-localization of PPM1G to the cytoplasm where the phosphatase can access a discrete set of substrates. Further, we unveil α-catenin, a component of adherens junction, as a new substrate for the PPM1G-B56 phosphatase complex in the cytoplasm. B56δ-PPM1G dephosphorylates α-catenin at serine 641, which is necessary for the appropriate assembly of adherens junctions and the prevention of aberrant cell migration. Collectively, we reveal a new holoenzyme with PPM1G-B56δ as integral components, in which the regulatory subunit provides accessibility to distinct substrates for the phosphatase by defining its cellular localization.

CRL7SMU1 E3 ligase complex-driven H2B ubiquitylation functions in sister chromatid cohesion by regulating SMC1 expression.

Cullin-RING-type E3 ligases (CRLs) control a broad range of biological processes by ubiquitylating numerous cellular substrates. However, the role of CRL E3 ligases in chromatid cohesion is unknown. In this study, we identified a new CRL-type E3 ligase (designated as CRL7SMU1 complex) that has an essential role in the maintenance of chromatid cohesion. We demonstrate that SMU1, DDB1, CUL7 and RNF40 are integral components of this complex. SMU1, by acting as a substrate recognition module, binds to H2B and mediates monoubiquitylation at the lysine (K) residue K120 through CRL7SMU1 E3 ligase complex. Depletion of CRL7SMU1 leads to loss of H2B ubiquitylation at the SMC1a locus and, thus, subsequently compromised SMC1a expression in cells. Knockdown of CRL7SMU1 components or loss of H2B ubiquitylation leads to defective sister chromatid cohesion, which is rescued by restoration of SMC1a expression. Together, our results unveil an important role of CRL7SMU1 E3 ligase in promoting H2B ubiquitylation for maintenance of sister chromatid cohesion during mitosis.This article has an associated First Person interview with the first author of the paper.

ERK1/2 activated PHLPP1 induces skeletal muscle ER stress through the inhibition of a novel substrate AMPK.

Nutritional abundance associated with chronic inflammation and dyslipidemia impairs the functioning of endoplasmic reticulum (ER) thereby hampering cellular responses to insulin. PHLPP1 was identified as a phosphatase which inactivates Akt, the master regulator of insulin mediated glucose homeostasis. Given the suggestive role of PHLPP1 phosphatase in terminating insulin signalling pathways, deeper insights into its functional role in inducing insulin resistance are warranted. Here, we show that PHLPP1 expression is enhanced in skeletal muscle of insulin resistant rodents which also displayed ER stress, an important mediator of insulin resistance. Using cultured cells and PHLPP1 knockdown mice, we demonstrate that PHLPP1 facilitates the development of ER stress. Importantly, shRNA mediated ablation of PHLPP1 significantly improved glucose clearance from systemic circulation with enhanced expression of glucose transporter 4 (GLUT-4) in skeletal muscle. Mechanistically, we show that endogenous PHLPP1 but not PP2Cα interacts with and directly dephosphorylates AMPK Thr172 in myoblasts without influencing its upstream kinase, LKB1. While the association between endogenous PHLPP1 and AMPK was enhanced in ER stressed cultured cells and soleus muscle of high fat diet fed mice, the basal interaction between PP2Ac and AMPK was minimally altered. Further, we show that PHLPP1α is phosphorylated by ERK1/2 at Ser932 under ER stress which is required for its ability to interact with and dephosphorylate AMPK and thereby induce ER stress. Taken together, our data position PHLPP1 as a key regulator of ER stress.

Post translational modifications of Rab GTPases.

Rab GTPases, the highly conserved members of Ras GTPase superfamily are central players in the vesicular trafficking. They are critically involved in intracellular trafficking pathway, beginning from formation of vesicles on donor membranes, defining trafficking specificity to facilitating vesicle docking on target membranes. Given the dynamic roles of Rabs during different stages of vesicular trafficking, mechanisms for their spatial and temporal regulation are crucial for normal cellular function. Regulation of Rab GTPase activity, localization and function has always been focused in and around the association of GDP dissociation inhibitor (GDI), Guanine nucleotide Exchange Factor (GEFs) and GTPase accelerating protein (GAP) to Rabs. However, several recent studies have highlighted the importance of different post-translational modifications in regulation of Rab activation and function. This review provides a summary of various post translational modifications (PTMs) and their significance to regulate localization and function of different Rabs.

PTEN Regulates Glucose Transporter Recycling by Impairing SNX27 Retromer Assembly.

The tumor suppressor PTEN executes cellular functions predominantly through its phosphatase activity. Here we identified a phosphatase-independent role for PTEN during vesicular trafficking of the glucose transporter GLUT1. PTEN physically interacts with SNX27, a component of the retromer complex that recycles transmembrane receptors such as GLUT1 from endosomes to the plasma membrane. PTEN binding with SNX27 prevents GLUT1 accumulation at the plasma membrane because of defective recycling and thus reduces cellular glucose uptake. Mechanistically, PTEN blocks the association of SNX27 with VPS26 and thereby hinders assembly of a functional retromer complex during the receptor recycling process. Importantly, we found a PTEN somatic mutation (T401I) that is defective in disrupting the association between SNX27 and VPS26, suggesting a critical role for PTEN in controlling optimal GLUT1 levels at the membrane to prevent tumor progression. Together, our results reveal a fundamental role of PTEN in the regulation of the SNX27 retromer pathway, which governs glucose transport and might contribute to PTEN tumor suppressor function.

A Human Tyrosine Phosphatase Interactome Mapped by Proteomic Profiling.

Tyrosine phosphatases play a critical role in many cellular processes and pathogenesis, yet comprehensive analysis of their functional interacting proteins in the cell is limited. By utilizing a proteomic approach, here we present an interaction network of 81 human tyrosine phosphatases built on 1884 high-confidence interactions of which 85% are unreported. Our analysis has linked several phosphatases with new cellular processes and unveiled protein interactions genetically linked to various human diseases including cancer. We validated the functional importance of an identified interaction network by characterizing a distinct novel interaction between PTPN5 and Mob1a. PTPN5 dephosphorylates Mob1a at Y26 residue. Further, we identify that PTPN5 is required for proper midbody abscission during cytokinesis through regulation of Mob1a dephosphorylation. In conclusion, our study provides a valuable resource of tyrosine phosphatase interactions, which can be further utilized to dissect novel cellular functions of these enzymes.

Interplay between the phosphatase PHLPP1 and E3 ligase RNF41 stimulates proper kinetochore assembly via the outer-kinetochore protein SGT1.

Kinetochores link chromosomes to spindle microtubules and are essential for accurate chromosome segregation during cell division. Kinetochores assemble at the centromeric region of chromosomes as a multiprotein complex. However, the molecular mechanisms of kinetochore assembly have not yet been fully elucidated. In this study, we identified pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) as a regulatory phosphatase that facilitates proper kinetochore assembly. We found that PHLPP1 interacted with the essential outer-kinetochore protein SGT1 and stabilized its protein levels. Loss of PHLPP1 from cells led to SGT1 degradation and thereby caused defective kinetochore assembly. We also found that the ring finger protein 41 (RNF41) as an E3 ligase ubiquitinated and degraded SGT1 in a phosphorylation-dependent manner. PHLPP1 dephosphorylated SGT1 at four conserved residues (Ser-17, Ser-249, Ser-289, and Thr-233) and thereby prevented SGT1 from associating with RNF41, in turn, countering SGT1 degradation. Importantly, depletion of RNF41 or expression of a non-phosphorylatable SGT1 mutant rescued the kinetochore defects caused by the loss of PHLPP1. Taken together, our results suggest that PHLPP1 plays an important role in the assembly of kinetochores by counteracting RNF41-mediated SGT1 degradation.

GINS complex protein Sld5 recruits SIK1 to activate MCM helicase during DNA replication.

In eukaryotes, proper loading and activation of MCM helicase at chromosomal origins plays a central role in DNA replication. Activation of MCM helicase requires its association with CDC45-GINS complex, but the mechanism of how this complex activates MCM helicase is poorly understood. Here we identified SIK1 (salt-inducible kinase 1), an AMPK related protein kinase, as a molecular link that connects GINS complex with MCM helicase activity. We demonstrated that Sld5 a component of GINS complex interacts with SIK1 and recruits it to the sites of DNA replication at the onset of S phase. Depletion of SIK1 leads to defective DNA replication. Further, we showed that SIK1 phosphorylates MCM2 at five conserved residues at its N-terminus, which is essential for the activation of MCM helicase. Collectively, our results suggest SIK1 as a novel integral component of CMG replicative helicase during eukaryotic DNA replication.

14-3-3σ Gene Loss Leads to Activation of the Epithelial to Mesenchymal Transition Due to the Stabilization of c-Jun Protein.

Loss of 14-3-3σ has been observed in multiple tumor types; however, the mechanisms by which 14-3-3σ loss leads to tumor progression are not understood. The experiments in this report demonstrate that loss of 14-3-3σ leads to a decrease in the expression of epithelial markers and an increase in the expression of mesenchymal markers, which is indicative of an induction of the epithelial to mesenchymal transition (EMT). The EMT was accompanied by an increase in migration and invasion in the 14-3-3σ(-/-) cells. 14-3-3σ(-/-) cells show increased stabilization of c-Jun, resulting in an increase in the expression of the EMT transcription factor slug. 14-3-3σ induces the ubiquitination and degradation of c-Jun in an FBW7-dependent manner. c-Jun ubiquitination is dependent on the presence of an intact nuclear export pathway as c-Jun is stabilized and localized to the nucleus in the presence of a nuclear export inhibitor. Furthermore, the absence of 14-3-3σ leads to the nuclear accumulation and stabilization of c-Jun, suggesting that 14-3-3σ regulates the subcellular localization of c-Jun. Our results have identified a novel mechanism by which 14-3-3σ maintains the epithelial phenotype by inhibiting EMT and suggest that this property of 14-3-3σ might contribute to its function as a tumor suppressor gene.

A modification switch on a molecular switch: Phosphoregulation of Rab7 during endosome maturation.

Rab GTPases, the highly conserved members of Ras GTPase superfamily are the pivotal regulators of vesicle-mediated trafficking. Rab GTPases, each with a specific subcellular localization, exert tremendous control over various aspects of vesicular transport, identity and dynamics. Several lines of research have established that GDI, GEFs and GAPs are the critical players to orchestrate Rab GTPase activity and function. The importance of post translational modifications in Rab GTPase functional regulation is poorly or not yet been addressed except for prenylation. Our recent study has revealed a novel dephosphorylation dependent regulatory mechanism for Rab7 activity and function. We have shown the importance of PTEN mediated dephosphorylation of Rab7 on highly conserved S72 and Y183 residues, which is essential for its GDI mediated membrane targeting and further activation by GEF. In conclusion, our study highlighted the importance of a phosphorylation/dephosphorylation switch in controlling timely Rab7 localization and activity on endosomes.

PTEN modulates EGFR late endocytic trafficking and degradation by dephosphorylating Rab7.

Tumour suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid phosphatase that negatively regulates growth factor-induced survival signalling. Here, we demonstrate that PTEN attenuates epidermal growth factor receptor (EGFR) signalling by promoting late endosome maturation by virtue of its protein phosphatase activity. Loss of PTEN impairs the transition of ligand-bound EGFR from early to late endosomes. We unveil Rab7, a critical GTPase for endosome maturation, as a functional PTEN interacting partner. PTEN dephosphorylates Rab7 on two conserved residues S72 and Y183, which are necessary for GDP dissociation inhibitor (GDI)-dependent recruitment of Rab7 on to late endosomes and subsequent maturation. Thus, our findings reveal PTEN-dependent endosome maturation through phosphoregulation of Rab7 as an important route of controlling EGFR signalling.

HACE1 mediated K27 ubiquitin linkage leads to YB-1 protein secretion.

Ubiquitination is an important post-translational modification that is implicated in controlling almost every biological process by targeting cellular proteins to degradation. While the importance of ubiquitination in controlling the fate and the intracellular functions of various proteins was widely studied, its role in extracellular protein secretion has been unexplored so far. In this study, by using YB-1 (Y-box Binding protein 1) as a model protein, we showed that ubiquitination is required for its extracellular secretion. We also identified HACE1 as a specific E3 ligase that polyubiquitinates YB-1 through non-canonical K27 linked ubiquitin chains. Formation of these ubiquitin linkages on YB-1 is necessary for its interaction with Tumor Susceptibility Gene 101 (TSG101), a component of the Multi-Vesicular Body (MVB) pathway, which facilitates its secretion. Finally, we demonstrated that extracellular secreted YB-1 is a functional protein that acts to inhibit Transforming Growth Factor-Beta mediated epithelial to mesenchymal transition. In summary, we identified a novel functional role for non-canonical ubiquitin linkages in mediating protein secretion.

The miRNA miR-34a enhances HIV-1 replication by targeting PNUTS/PPP1R10, which negatively regulates HIV-1 transcriptional complex formation.

HIV-1 relies heavily on the host cellular machinery for its replication. During infection, HIV-1 is known to modulate the host-cell miRNA profile. One of the miRNAs, miR-34a, is up-regulated by HIV-1 in T-cells as suggested by miRNA microarray studies. However, the functional consequences and the mechanism behind this phenomenon were not explored. The present study shows that HIV-1 enhances miR-34a in a time-dependent manner in T-cells. Our overexpression and knockdown-based experimental results suggest that miR-34a promotes HIV-1 replication in T-cells. Hence, there is a positive feedback loop between miR-34a and HIV-1 replication. We show that the mechanism of action of miR-34a in HIV-1 replication involves a cellular protein, the phosphatase 1 nuclear-targeting subunit (PNUTS). PNUTS expression levels decrease with the progression of HIV-1 infection in T-cells. Also, the overexpression of PNUTS potently inhibits HIV-1 replication in a dose-dependent manner. We report for the first time that PNUTS negatively regulates HIV-1 transcription by inhibiting the assembly of core components of the transcription elongation factor P-TEFb, i.e. cyclin T1 and CDK9. Thus, HIV-1 increases miR-34a expression in cells to overcome the inhibitory effect of PNUTS on HIV-1 transcription. So, the present study provides new mechanistic details with regard to our understanding of a complex interplay between miR-34a and the HIV-1 transcription machinery involving PNUTS.

Mapping of apoptin-interaction with BCR-ABL1, and development of apoptin-based targeted therapy.

Majority of chronic myeloid leukemia patients experience an adequate therapeutic effect from imatinib however, 26-37% of patients discontinue imatinib therapy due to a suboptimal response or intolerance. Here we investigated derivatives of apoptin, a chicken anemia viral protein with selective toxicity towards cancer cells, which can be directed towards inhibiting multiple hyperactive kinases including BCR-ABL1. Our earlier studies revealed that a proline-rich segment of apoptin interacts with the SH3 domain of fusion protein BCR-ABL1 (p210) and acts as a negative regulator of BCR-ABL1 kinase and its downstream targets. In this study we show for the first time, the therapeutic potential of apoptin-derived decapeptide for the treatment of CML by establishing the minimal region of apoptin interaction domain with BCR-ABL1. We further show that the apoptin decapeptide is able to inhibit BCR-ABL1 down stream target c-Myc with a comparable efficacy to full-length apoptin and Imatinib. The synthetic apoptin is able to inhibit cell proliferation in murine (32Dp210), human cell line (K562), and ex vivo in both imatinib-resistant and imatinib sensitive CML patient samples. The apoptin based single or combination therapy may be an additional option in CML treatment and eventually be feasible as curative therapy.

WWP2-WWP1 ubiquitin ligase complex coordinated by PPM1G maintains the balance between cellular p73 and ΔNp73 levels.

The balance between transcription factor p73 and its functionally opposing N-terminally truncated ΔNp73 isoform is critical for cell survival, but the precise mechanism that regulates their levels is not clear. In our study, we identified WWP2, an E3 ligase, as a novel p73-associated protein that ubiquitinates and degrades p73. In contrast, WWP2 heterodimerizes with another E3 ligase, WWP1, which specifically ubiquitinates and degrades ΔNp73. Further, we identified phosphatase PPM1G as a functional switch that controls the balance between monomeric WWP2 and a WWP2/WWP1 heterodimeric state in the cell. During cellular stress, WWP2 is inactivated, leading to upregulation of p73, whereas WWP2-WWP1 complex is intact to degrade ΔNp73, thus playing an important role in shifting the balance between p73 and ΔNp73. Collectively, our results reveal a new functional E3 ligase complex controlled by PPM1G that differentially regulates cellular p73 and ΔNp73.

Deubiquitylation and stabilization of PTEN by USP13.

The tumour suppressor PTEN is frequently lost in human cancers. In addition to gene mutations and deletions, recent studies have revealed the importance of post-translational modifications, such as ubiquitylation, in the regulation of PTEN stability, activity and localization. However, the deubiquitylase that regulates PTEN polyubiquitylation and protein stability remains unknown. Here we screened a total of 30 deubiquitylating enzymes (DUBs) and identified five DUBs that physically associate with PTEN. One of them, USP13, stabilizes the PTEN protein through direct binding and deubiquitylation of PTEN. Loss of USP13 in breast cancer cells promotes AKT phosphorylation, cell proliferation, anchorage-independent growth, glycolysis and tumour growth through downregulation of PTEN. Conversely, overexpression of USP13 suppresses tumorigenesis and glycolysis in PTEN-positive but not PTEN-null breast cancer cells. Importantly, USP13 protein is downregulated in human breast tumours and correlates with PTEN protein levels. These findings identify USP13 as a tumour-suppressing protein that functions through deubiquitylation and stabilization of PTEN.