Ali Sarkeshik

PYR/PYL/RCAR family members are major in-vivo ABI1 protein phosphatase 2C-interacting proteins in Arabidopsis

Abscisic acid (ABA) mediates resistance to abiotic stress and controls developmental processes in plants. The group-A PP2Cs, of which ABI1 is the prototypical member, are protein phosphatases that play critical roles as negative regulators very early in ABA signal transduction. Because redundancy is thought to limit the genetic dissection of early ABA signalling, to identify redundant and early ABA signalling proteins, we pursued a proteomics approach. We generated YFP-tagged ABI1 Arabidopsis expression lines and identified in vivo ABI1-interacting proteins by mass-spectrometric analyses of ABI1 complexes. Known ABA signalling components were isolated including SnRK2 protein kinases. We confirm previous studies in yeast and now show that ABI1 interacts with the ABA-signalling kinases OST1, SnRK2.2 and SnRK2.3 in plants. Interestingly, the most robust in planta ABI1-interacting proteins in all LC-MS/MS experiments were nine of the 14 PYR/PYL/RCAR proteins, which were recently reported as ABA-binding signal transduction proteins, providing evidence for in vivo PYR/PYL/RCAR interactions with ABI1 in Arabidopsis. ABI1–PYR1 interaction was stimulated within 5 min of ABA treatment in Arabidopsis. Interestingly, in contrast, PYR1 and SnRK2.3 co-immunoprecipitated equally well in the presence and absence of ABA. To investigate the biological relevance of the PYR/PYLs, we analysed pyr1/pyl1/pyl2/pyl4 quadruple mutant plants and found strong insensitivities in ABA-induced stomatal closure and ABA-inhibition of stomatal opening. These findings demonstrate that ABI1 can interact with several PYR/PYL/RCAR family members in Arabidopsis, that PYR1–ABI1 interaction is rapidly stimulated by ABA in Arabidopsis and indicate new SnRK2 kinase-PYR/PYL/RCAR interactions in an emerging model for PYR/PYL/RCAR-mediated ABA signalling.

Angiomotin family proteins are novel activators of the LATS2 kinase tumor suppressor

The Hippo pathway kinase LATS2 promotes contact inhibition of growth. How LATS2 is activated in response to changes in cell density is unknown. It is found that tight junction protein AMOTL2 is a novel activator of LATS2, raising the possibility that tight junction assembly promotes LATS2-dependent inhibition of cell proliferation.

TFG-1 function in protein secretion and oncogenesis

Export of proteins from the endoplasmic reticulum in COPII-coated vesicles occurs at defined sites that contain the scaffolding protein Sec16. We identify TFG-1, a new conserved regulator of protein secretion that interacts directly with SEC-16 and controls the export of cargoes from the endoplasmic reticulum in Caenorhabditis elegans. Hydrodynamic studies indicate that TFG-1 forms hexamers that facilitate the co-assembly of SEC-16 with COPII subunits. Consistent with these findings, TFG-1 depletion leads to a marked decline in both SEC-16 and COPII levels at endoplasmic reticulum exit sites. The sequence encoding the amino terminus of human TFG has been previously identified in chromosome translocation events involving two protein kinases, which created a pair of oncogenes. We propose that fusion of these kinases to TFG relocalizes their activities to endoplasmic reticulum exit sites, where they prematurely phosphorylate substrates during endoplasmic reticulum export. Our findings provide a mechanism by which translocations involving TFG can result in cellular transformation and oncogenesis.Export of proteins from the endoplasmic reticulum in COPII-coated vesicles occurs at defined sites that contain the scaffolding protein Sec16. We identify TFG-1, a new conserved regulator of protein secretion that interacts directly with SEC-16 and controls the export of cargoes from the endoplasmic reticulum in Caenorhabditis elegans. Hydrodynamic studies indicate that TFG-1 forms hexamers that facilitate the co-assembly of SEC-16 with COPII subunits. Consistent with these findings, TFG-1 depletion leads to a marked decline in both SEC-16 and COPII levels at endoplasmic reticulum exit sites. The sequence encoding the amino terminus of human TFG has been previously identified in chromosome translocation events involving two protein kinases, which created a pair of oncogenes. We propose that fusion of these kinases to TFG relocalizes their activities to endoplasmic reticulum exit sites, where they prematurely phosphorylate substrates during endoplasmic reticulum export. Our findings provide a mechanism by which translocations involving TFG can result in cellular transformation and oncogenesis.

Role of Oxysterol Binding Protein in Hepatitis C Virus infection

ABSTRACT Hepatitis C virus (HCV) RNA genome replicates within the ribonucleoprotein (RNP) complex in the modified membranous structures extended from endoplasmic reticulum. A proteomic analysis of HCV RNP complexes revealed the association of oxysterol binding protein (OSBP) as one of the components of these complexes. OSBP interacted with the N-terminal domain I of the HCV NS5A protein and colocalized to the Golgi compartment with NS5A. An OSBP-specific short hairpin RNA that partially downregulated OSBP expression resulted in a decrease of the HCV particle release in culture supernatant with little effect on viral RNA replication. The pleckstrin homology (PH) domain located in the N-terminal region of OSBP targeted this protein to the Golgi apparatus. OSBP deletion mutation in the PH (ΔPH) domain failed to localize to the Golgi apparatus and inhibited the HCV particle release. These studies suggest a possible functional role of OSBP in the HCV maturation process.

PRMT1 Interacts with AML1-ETO to Promote Its Transcriptional Activation and Progenitor Cell Proliferative Potential

Fusion protein AML1-ETO, resulting from t(8;21) translocation, is highly related to leukemia development. It has been reported that full-length AML1-ETO blocks AML1 function and requires additional mutagenic events to promote leukemia. We have previously shown that the expression of AE9a, a splice isoform of AML1-ETO, can rapidly cause leukemia in mice. To understand how AML1-ETO is involved in leukemia development, we took advantage of our AE9a leukemia model and sought to identify its interacting proteins from primary leukemic cells. Here, we report the discovery of a novel AE9a binding partner PRMT1 (protein arginine methyltransferase 1). PRMT1 not only interacts with but also weakly methylates arginine 142 of AE9a. Knockdown of PRMT1 affects expression of a specific group of AE9a-activated genes. We also show that AE9a recruits PRMT1 to promoters of AE9a-activated genes, resulting in enrichment of H4 arginine 3 methylation, H3 Lys9/14 acetylation, and transcription activation. More importantly, knockdown of PRMT1 suppresses the self-renewal capability of AE9a, suggesting a potential role of PRMT1 in regulating leukemia development.

Reconstitution and Protein Composition Analysis of Endocytic Actin Patches

BACKGROUND Clathrin-actin-mediated endocytosis in yeast involves the progressive assembly of at least 60 different proteins at cortical sites. More than half of these proteins are involved in the assembly of a branched network of actin filaments to provide the forces required for plasma membrane invagination. RESULTS To gain insights into the regulation of endocytic actin patch dynamics, we developed an in vitro actin assembly assay using microbeads functionalized with the nucleation promoting factor (NPF) Las17 (yeast WASP). When incubated in a yeast extract, these beads assembled actin networks, and a significant fraction became motile. Multidimensional protein identification technology (MudPIT) showed that the recruitment of actin-binding proteins to these Las17-derived actin networks is selective. None of the proteins known to exclusively regulate the in vivo formation of actin cables or the actin contractile ring were identified. Our analysis also identified components of three other cortical structures, eisosomes, phosphoinositide kinase (PIK) patches, and the TORC2 complex, establishing intriguing biochemical connections between four different yeast cortical complexes. Finally, we identified Aim3 as a regulator of actin dynamics at endocytic sites. CONCLUSIONS WASP is sufficient to trigger assembly of actin networks composed selectively of actin patch proteins. These experiments establish that the protein composition of different F-actin structures is determined by the protein factor that initiates the network. The identification of binding partners revealed new biochemical connections between WASP-derived networks and other cortical complexes and identified Aim3 as a novel regulator of the endocytic actin patch.

Reptin and Pontin function antagonistically with PcG and TrxG complexes to mediate Hox gene control

Pontin (Pont) and Reptin (Rept) are paralogous ATPases that are evolutionarily conserved from yeast to human. They are recruited in multiprotein complexes that function in various aspects of DNA metabolism. They are essential for viability and have antagonistic roles in tissue growth, cell signalling and regulation of the tumour metastasis suppressor gene, KAI1, indicating that the balance of Pont and Rept regulates epigenetic programmes critical for development and cancer progression. Here, we describe Pont and Rept as antagonistic mediators of Drosophila Hox gene transcription, functioning with Polycomb group (PcG) and Trithorax group proteins to maintain correct patterns of expression. We show that Rept is a component of the PRC1 PcG complex, whereas Pont purifies with the Brahma complex. Furthermore, the enzymatic functions of Rept and Pont are indispensable for maintaining Hox gene expression states, highlighting the importance of these two antagonistic factors in transcriptional output.

Reduced pachytene piRNAs and translation underlie spermiogenic arrest in Maelstrom mutant mice

Pachytene piRNAs are a class of Piwi‐interacting small RNAs abundant in spermatids of the adult mouse testis. They are processed from piRNA primary transcripts by a poorly understood mechanism and, unlike fetal transposon‐derived piRNAs, lack complementary targets in the spermatid transcriptome. We report that immunopurified complexes of a conserved piRNA pathway protein Maelstrom (MAEL) are enriched in MIWI (Piwi partner of pachytene piRNAs), Tudor‐domain proteins and processing intermediates of pachytene piRNA primary transcripts. We provide evidence of functional significance of these complexes in Mael129 knockout mice that exhibit spermiogenic arrest with acrosome and flagellum malformation. Mael129‐null mutant testes possess low levels of piRNAs derived from MAEL‐associated piRNA precursors and exhibit reduced translation of numerous spermiogenic mRNAs including those encoding acrosome and flagellum proteins. These translation defects in haploid round spermatids are likely indirect, as neither MAEL nor piRNA precursors associate with polyribosomes, and they may arise from an imbalance between pachytene piRNAs and MIWI.

Mitotic Spindle Proteomics in Chinese Hamster Ovary Cells

Mitosis is a fundamental process in the development of all organisms. The mitotic spindle guides the cell through mitosis as it mediates the segregation of chromosomes, the orientation of the cleavage furrow, and the progression of cell division. Birth defects and tissue-specific cancers often result from abnormalities in mitotic events. Here, we report a proteomic study of the mitotic spindle from Chinese Hamster Ovary (CHO) cells. Four different isolations of metaphase spindles were subjected to Multi-dimensional Protein Identification Technology (MudPIT) analysis and tandem mass spectrometry. We identified 1155 proteins and used Gene Ontology (GO) analysis to categorize proteins into cellular component groups. We then compared our data to the previously published CHO midbody proteome and identified proteins that are unique to the CHO spindle. Our data represent the first mitotic spindle proteome in CHO cells, which augments the list of mitotic spindle components from mammalian cells.

Regulation of ubiquitin-dependent cargo sorting by multiple endocytic adaptors at the plasma membrane

Endocytic protein trafficking is directed by sorting signals on cargo molecules that are recognized by cytosolic adaptor proteins. However, the steps necessary to segregate the variety of cargoes during endocytosis remain poorly defined. Using Caenorhabditis elegans, we demonstrate that multiple plasma membrane endocytic adaptors function redundantly to regulate clathrin-mediated endocytosis and to recruit components of the endosomal sorting complex required for transport (ESCRT) machinery to the cell surface to direct the sorting of ubiquitin-modified substrates. Moreover, our data suggest that preassembly of cargoes with the ESCRT-0 complex at the plasma membrane enhances the efficiency of downstream sorting events in the endolysosomal system. In the absence of a heterooligomeric adaptor complex composed of FCHO, Eps15, and intersectin, ESCRT-0 accumulation at the cell surface is diminished, and the degradation of a ubiquitin-modified cargo slows significantly without affecting the rate of its clathrin-mediated internalization. Consistent with a role for the ESCRT machinery during cargo endocytosis, we further show that the ESCRT-0 complex accumulates at a subset of clathrin-coated pits on the surface of human cells. Our findings suggest a unique mechanism by which ubiquitin-modified cargoes are sequestered into the endolysosomal pathway.