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PhD cell biologist

Gan HaDarom, Israel
December 29, 2019

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Dear Sir or Madam,

I would like to apply for Assistant Professor Faculty position at your Department. Few words about myself: I am Israeli citizen, currently live in Israel. I graduated with PhD in molecular cell neurobiology from The Weizmann Institute of Science, Israel, working on molecular composition and modulation of potassium channels in myelin-forming glial cells and their role in cell proliferation (lab of prof. Bernard Attali, now in Tel Aviv University). Later, I carried out successful postdoctoral studies (EMBO Long-Term Fellowship) at Cell and Developmental Biology, UC San Diego, working on ubiquitination and SUMOylation of MEK1 protein kinase in Dictyostelium cell migration (lab of prof. Richard A. Firtel). I also completed senior internship in cellular senescence at the Dept. of Molecular Cell Biology, Weizmann Institute of Science (lab of prof. Valery Krizhanovsky), where I carried out genome-wide RNAi viability screen in drug-induced senescent cells.

Attached, to this my letter, please, find my CV, including list of publications, Research Summary and contact information of my referees.

I am very excited by the opportunity to contribute to research work and teaching at the Department. I am looking forward to hearing from you!


Alexander Sobko, PhD


Curriculum Vitae

Alex Sobko, PhD




Websites: - me - alexander/


• International research experience (Canada, USA, Israel); • Strong skills in cell signaling, applied genetics and molecular cell systems biology; neurobiology; • More than 5 years of industry biotech and academic research (Staff Scientist), more than 3 years of academic postdoctoral work in cell and developmental biology; • Good analytical skills and thorough understanding of biological factors that influence gene and protein expression; practical skills in establishing new assays to detect protein- protein interactions, posttranslational protein modifications, correlations between biomarker expression, growth, and cellular structures; cell-based in vitro and in vivo biochemical assays to determine molecular identity, expression, activities, and localization of signaling enzymes, biomarkers; quantitative analysis of subcellular protein and organelle localization, gene and protein expression; experimental design, data mining and analysis of proteomics and transcriptomics experiments; • Applied bioinformatics tools to interpret gene and protein sequence information, genomic databases, protein and DNA modifications software for reconstruction of protein-protein interactions, cell signaling pathways; • Excellent record of project and research group management, collaborations, contribution to peer-reviewed research publications, progress reports, publication quality presentations at professional conferences; supervisory and professional review in matrix organization


Work Experience


Research of biological mechanisms of cellular senescence. Conducted cell biology studies of drug- induced senescence; conducted RNAi viability screen in drug-induced senescent cells. STAFF SCIENTIST, IOGEN CORPORATION Ottawa, Ontario, Canada 2003-2007 Research of biological mechanisms affecting productivity of industrial microorganisms; optimization of industrial performance; determined intrinsic correlation between fermentation productivity and cell growth parameters, such as activities of nutrient- and stress-sensitive protein kinases, cell size and cell cycle progression; analyzed functions of unfolded protein response, degradation and aggregation of cellulase enzymes, contribution of these factors to fermentation productivity; developed and applied real-time qPCR-based assays to measure expression of enzymes and biomarkers of growth, viability, secretion, environmental stress responses in production microorganisms, established imaging-based assays to examine colony growth, cell morphology, metabolic biomarkers; contributed to collaborative EST DNA microarrays work to measure gene expression on genomic scale, real-time PCR; contributed to and authored presentations at international conferences, authored a comprehensive peer-review article on the control of gene and protein expression, cell growth, stress responses, chronological and replicative cell aging published in Science Signaling Magazine -Signal Transduction Knowledge Environment (STKE); obtained corporate training in business communication skills program SPECIALIST, UNIVERSITY OF CALIFORNIA, DIVISION OF BIOLOGICAL SCIENCES, San Diego, California, USA 2001-2003

Research in molecular genetics of chemotaxis and protein kinase signaling in Dictyostelium; time- lapse and fluorescent microscopy-based assays to study cell migration and dynamic localization of signaling proteins in response to stimulation of chemoattractant receptors; reconstructions and quantitative analyses of protein and organelle localization, established and validated the assays to measure enzymatic activities (protein kinases; ubiquitin ligase), actin cytoskeleton dynamics, worked with genomics databases, cloned mRNAs for expression in genetically tractable cell lines for imaging/biochemical assays; sucessfully contributed as first author to peer-reviewed article and presentations (Sobko A., Developmental Cell, 2002). Education

POSTGRADUATE RESEARCH BIOLOGIST, UNIVERSITY OF CALIFORNIA, DIVISION OF BIOLOGICAL SCIENCES, San Diego, California, USA 1999-2001 EMBO Long-term Postdoctoral Fellowship in Cell & Developmental Biology Training in cell signaling, protein modifications, subcellular localization of signaling proteins, regulated proteolysis, imaging of cell migration, dynamics, analyses of complex cell populations, functional genomics and proteomics.


PhD Neurobiology, 1995-2000

Thesis: Sobko A. (1999). Voltage-gated potassium channels in myelin-forming glial cells and glial progenitors: molecular structure, developmental expression and role in cell proliferation. 4

Course work: molecular, cellular and integrative neuroscience, cell signal transduction, developmental neurobiology, protein chemistry, biophysics, molecular biology and genetics of receptors and ion channels.


MSc Neurobiology, With Distinction

Thesis: Sobko A. (1994). The regulation of glucocorticoid binding in lymphoid tissues and brain and its role in bidirectional communication between the central nervous and immune systems. Course work: molecular and cellular neurobiology, cell biology of exocytosis and secretion, neuroendocrinology, neuroimmunology.

Supplemental Professional Training and Presentations IOGEN Business Communication Skills Training, Ottawa, Ontario, 2006 Gordon Research Conference, New Port, RI, 2005

Stress Proteins in Growth, Development and Disease 7-th European Conference on Fungal Genetics, Copenhagen, Denmark, 2004 Basic and Industrial Microbiology, Fungal Genomics 1-st International Conference on Ubiquitin-like Modifications, MD Anderson Center, Texas, 2002

International Dictyostelium Conference, San Diego, CA, 2000 American Society for Neuroscience Meeting, Los Angeles, CA 1998 Israeli Society for Neuroscience Annual Meeting, Eilat, Israel, 1998 Prizes, Distinctions, Grants and Awards

EMBO Long-term Postdoctoral Fellowship, 1999-2001

Weizmann Institute Feinberg Graduate School Scholarship, 1995-1999 Hebrew University Medical School Prize for Excellence in M.Sc. Thesis Hebrew University Graduate Scholarship in Biomedical Sciences 5


Sobko A. Systems biology of AGC kinases in fungi. Science STKE. 2006 Sep 12;2006(352):re9. Review. PMID: 16971477.

Sobko A, Ma H, Firtel RA. Regulated SUMOylation and ubiquitination of DdMEK1 is required for proper chemotaxis. Dev Cell. 2002 Jun;2(6):745-56. PubMed PMID: 12062087. Soliven B, Ma L, Bae H, Attali B, Sobko A, Iwase T. PDGF upregulates delayed rectifier via Src family kinases and sphingosine kinase in oligodendroglial progenitors. Am J Physiol Cell Physiol. 2003 Jan;284(1):C85-93. PMID: 12475761.

Levite M, Cahalon L, Peretz A, Hershkoviz R, Sobko A, Ariel A, Desai R, Attali B, Lider O. Extracellular K and opening of voltage-gated potassium channels activate T cell integrin function: physical and functional association between Kv1.3 channels and beta1 integrins. J Exp Med. 2000 Apr 3;191(7):1167-76. PubMed PMID: 10748234; PMCID: PMC2193178. Peretz A, Gil-Henn H, Sobko A, Shinder V, Attali B, Elson A. Hypomyelination and increased activity of voltage-gated K channels in mice lacking protein tyrosine phosphatase epsilon. EMBO J. 2000 Aug 1;19(15):4036-45. PubMed PMID: 10921884; PMCID: PMC306594. Peretz A, Sobko A, Attali B. Tyrosine kinases modulate K+ channel gating in mouse Schwann cells. J Physiol. 1999 Sep 1;519 Pt 2:373-84. PubMed PMID: 10457056; PMCID: PMC2269503. Peretz A, Abitbol I, Sobko A, Wu CF, Attali B. A Ca2+/calmodulin-dependent protein kinase modulates Drosophila photoreceptor K+ currents: a role in shaping the photoreceptor potential. J Neurosci. 1998 Nov 15;18(22):9153-62. PMID: 9801355. Sobko A, Peretz A, Shirihai O, Etkin S, Cherepanova V, Dagan D, Attali B. Heteromultimeric delayed-rectifier K+ channels in schwann cells: developmental expression and role in cell proliferation. J Neurosci. 1998 Dec 15;18(24):10398-408. PMID: 9852577. Sobko A, Peretz A, Attali B. Constitutive activation of delayed-rectifier potassium channels by a src family tyrosine kinase in Schwann cells. EMBO J. 1998 Aug 17; 17(16):4723-34. PMID: 9707431.

Attali B, Wang N, Kolot A, Sobko A, Cherepanov V, Soliven B. Characterization of delayed rectifier Kv channels in oligodendrocytes and progenitor cells. J Neurosci. 1997 Nov 1;17(21):8234-45. PMID: 9334399

Ovadia H, Sobko A, Wholmann A, Weidenfeld J. Cellular nuclear binding and retention of glucocorticoids in rat lymphoid cells: effect of long-term adrenalectomy. Neuroimmunomodulation. 1995 Nov-Dec;2(6):339-46. PMID: 8840336. 6



Metastasis remains the greatest challenge in the clinical management of cancer. Cell motility is fundamental and ancient cellular behavior that contributes to metastasis and is conserved in non- mammalian model organisms, such as Dictyostelium discoideum (34 MB sequenced genome; ~ 12,500 predicted proteins; ~ 295 protein kinases). Dictyostelium presents a simple model to study directed cell migration. It has been proposed that in their most invasive form, metastatic cancer cells revert to the primitive mode of amoeboid migration that is shared by haematopoietic cells and Dictyostelium. The evolutionary conservation of chemokine signalling pathways, accessible genetics and amenability to live imaging make Dictyostelium an important model to examine basic molecular mechanisms that govern chemokine-mediated chemotaxis. This organism permits direct observation of cells moving in complex native environment and allows large-scale genetic and pharmacological screening, as well as extensive biochemical and cell biology studies. Below, we highlight insights derived from study of Dictyostelium, including the detailed signaling network, which governs chemotaxis. While the motility of tumor cells and certain host cells promotes metastatic spread, the motility of tumor-reactive T-cells increases their anti-tumor effects. It is important to elucidate the mechanisms of cell motility, with the ultimate goal of identifying combination therapies that increase motility of beneficial cells and block the spread of harmful cells. Identifying the basic mechanisms that regulate chemotactic signaling may reveal how to increase the motility and invasion of tumor- reactive T cells into tumors. This is an important frontier in expanding the current success of immunotherapeutic agents, as a key limitation to the effectiveness of these treatments is the inability of T cells to infiltrate tumors. During nutrient deprivation, Dictyostelium cells chemotax towards secreted cAMP, leading to the formation of aggregates that differentiate into spore and stalk cells to form fruiting bodies. Chemotactic migration is initiated when cAMP binds to the G protein-coupled cAMP receptor 1. This leads to the dissociation of the G protein subunits, which then activate a variety of signaling cascades that converge to polarize the cell and initiate cell migration. The highly orchestrated rearrangements of actin and myosin cytoskeleton result in amoeboid migration. Similarly, chemokine- and growth factor-mediated chemotaxis was observed in tumors. Chemokines attract numerous tumor cell types. Also, immune cells, which chemotax to sites of inflammation and injury, are subverted by tumors to infiltrate tissue and support tumor growth via similar chemotactic mechanisms.

Use of green fluorescent protein (GFP) revealed that, whereas both cAR1 and its associated G protein remain uniformly distributed in chemotaxing cells, proteins harboring pleckstrin homology

(PH) domains that bind to phosphatidylinositol- 3,4,5-trisphosphate (PIP3) specifically redistribute to the leading edge of chemotaxing cells. This molecular underpinning of directed amoeboid migration was also observed in neutrophils and in fibroblasts in response to gradients of platelet-derived growth factor. The asymmetrical distribution of PIP3 results from the spatial activation of PI3K at the front and PTEN at the sides and rear of cells. These localized PIP3 signals therefore provide spatial orientation for PH domain-containing proteins that act as adaptors for specific downstream cascades, which, in the case of chemotaxis, spatially nucleate actin assembly. 7

In human cancers, mutations in the PI3K–PTEN–mTOR complex 1 cascade are prevalent, potentially dysregulating the PIP3 signals necessary for migration. PI3K–AKT signaling can increase invasion by up-regulating matrix metalloproteinase 9. Additionally, PI3K is a key regulator of immune cell migration as well as cellular processes such as cell growth, survival and differentiation. Thus, inhibitors of this pathway, which have been extensively studied to treat cancer, likely exert pleiotropic effects on host cancer-associated cells, such as immune cells and fibroblasts, in addition to affecting the tumor cells directly. Genetic screens are another powerful tool used to identify pathways controlling directed cell migration in Dictyostelium. In this way, several independent pathways parallel to the PI3K–PTEN signaling cascade were identified in genetic screens for migration defective mutants. Among these genetically dissected pathways, MAP kinase pathways are central in control of aggregation and chemotaxis.

MAP kinase kinase (DdMEK1) is required for proper aggregation in Dictyostelium. Null mutations produce extremely small aggregate sizes, resulting in the formation of slugs and terminal fruiting bodies that are significantly smaller than those of wild-type cells. Time-lapse video microscopy assays indicate that cells are unable to undergo chemotaxis properly during aggregation in response to cAMP or activate guanylyl cyclase, another known regulator of chemotaxis in Dictyostelium. Expression of putative constitutively active forms of DdMEK1 in a ddmek1 null background is capable, at least partially, of complementing the small aggregate size defect. The activation of the MAP kinase ERK2, which is essential for chemoattractant activation of adenylyl cyclase, is not affected in ddmek1 null strains, indicating that DdMEK1 does not regulate ERK2 and suggesting that at least two independent MAP kinase cascades control aggregation in Dictyostelium. Indeed, we found that DdMEK1 most likely activates ERK1. We elucidated a pathway regulating the localization and function of DdMEK1. DdMEK1 is rapidly and transiently SUMOylated in response to cAMP. SUMOylation is required for MEK1's function and its translocation from the nucleus to the cytosol and cortex, including the leading edge of chemotaxing cells. DdMEK1 in which the site of SUMOylation is mutated is retained in the nucleus and does not complement the mek1 null phenotype. Constitutively active DdMEK1 is cytosolic and is constitutively SUMOylated, whereas the corresponding non-activatable DdMEK1 is not SUMOylated and nuclear. MEK1 is also ubiquitinated in response to signaling. A MEK1- interacting, ubiquitin E3 ligase RING domain-containing protein controls nuclear localization and MEK1 ubiquitination. We studied the composition and function of this signaling complex that consists of DdMEK1 and associated DdMEK1-interacting protein (MIP). We found that MIP possesses RING domain that serves as functional ubiquitin ligase module, which ubiquitinates MEK1 in vivo and in vitro and targets this kinase protein for proteasome degradation. It is interesting yet unexplored whether DdMEK1 phosphorylates MIP and if so whether MIP ubiquitinates other unknown substrates. The identity of DdMEK1 SUMO Ligase is also unknown. It is plausible possibility that MIP is somehow involved in SUMOylation (Sobko A, Ma H, Firtel RA. Developmental Cell. 2002 Jun; 2(6):745-56).

Several years later, a Japanese group (Kubota, O’Grady, Saito, Takekawa, 2011, Nature Cell Biology) confirmed that MEK SUMOylation occurs also in mammalian cells, not only in Dictyostelium. This opens up the possibility, that MEK SUMOylation somehow also plays a role in other organisms as well. Therefore, it could be, in principle, possible to test this hypothesis by CRISPR gene editing – substituting 8

SUMOylation sites - lysine residues in genomic MEK with arginines in Dictyostelium and mice, in certain types of cells and test whether such transgenic organisms exhibit certain phenotypes. In my opinion, this could make exciting new project for research! I am also interested in the paradigms of recently discovered group of SUMO-targeted Ubiquitin Ligases (such as MIP, which we discovered in Dictyostelium). Especially exciting is the idea of using heterobifunctional small molecules, referred to as PROTACs, to tether cellular proteins to a ubiquitin ligase, resulting in Ubiquitination and degradation of the tethered protein.

I am also interested in the association of protein kinase B (AKT)-like kinase Sch9 with the proteins that participate in protein degradation elucidated, using S. cerevisiae. One of the interesting outcomes of the proteome-wide analysis of protein-protein interactions in yeast is the finding that Sch9 associates with Shp1, Cdc48, and Ufd1, which form a complex responsible for the recognition and targeting of ubiquitinated proteins to the proteasome for degradation (Sobko A Science Signaling, 2006). What is the result of the interaction of Sch9 with this complex of the protein-degradation machinery? One possibility is that Sch9 itself is ubiquitinated and targeted for degradation by Shp1-Cdc48-Ufd1. An alternative scenario is that Sch9 phosphorylates some of the components of this complex to regulate its activity. It is also interesting to investigate whether this multi-protein complex is evolutionarily conserved and could exist for other AKT/PKB homologues.

In my future research I am interested to explore composition and functions of other multi-protein signaling complexes that consist of protein kinases and associated proteins, in particular, those, that participate in proteasome degradation. Other components of DdMEK1 and Sch9 signaling complexes will be also explored. In addition, I am interested to study the homologues and functional counterparts of these protein kinases in human genome and to identify the functional DNA sequence variants of these proteins and their roles in cell biology and diseases, such as cancer.



Rationale and hypothesis:

In our previous study (Sobko A. et al, Dev Cell, 2002) we characterized SUMO targeted Ubiquitin Ligase (StUbL) MIP1 that associates with protein kinase MEK1 and targets SUMOylated MEK1 to ubiquitination and proteasomal degradation. These modifications happen in response to activation of MEK1 by chemoattractant cAMP. Another study from Firtel lab characterized SMEK

– second site genetic suppressor of mek1- null phenotype (Mendoza M et al., Mol Cell Biol. 2005). Deletion of mek1 gene results in the phenotype, in which cells fail to aggregate properly and form very small aggregates, due to severe chemotaxis defect. Suppressor phenotype of SMEK implies that aggregation/chemotaxis defect of mek1 null cells is rescued upon deletion of smek gene in mek1- null cells. According to Mendoza et al., the analysis of smek phenotype shows, that not all effects of SMEK occur via MEK1 signaling. Nevertheless, at least in part, MEK1 sends the signal to SMEK, which, in turn, negatively affects chemotaxis and aggregation. Therefore, we propose the following scenario: MEK1 and SMEK belong to the same linear pathway, in which MEK1 negatively regulates SMEK, which then negatively regulates chemotaxis and aggregation. 9

MIP1 is a RING Finger protein, which belongs to recently discovered evolutionarily conserved group of proteins, that contain also SUMO Interactive Motif (SIM) and drive SUMOylated proteins to ubiquitination and subsequent proteasomal degradation (Sun H. et al., EMBO J., 2007). RNF4 is mammalian homologue of MIP. It also contains SIM and RING Finger domains and it is most likely functions as StUbL. Recently, RNF4-interacting proteins were systematically identified in high throughput proteomics/mass spectrometry study (Kumar R. et al., Nature Commun., 2017). Intriguingly, the data of this study shows that RNF4 interacts with human homologue of Dictyostelium SMEK – hSMEK2. This raises the possibility, that such complex is conserved in evolution, and exists in both human and Dictytostelium cells. If in human cells RNF4 interacts with hSMEK2, then, MIP1 possibly interacts with SMEK in Dictyostelium. We propose that MEK1 and SMEK interact not only genetically, but also physically. Moreover, the mechanism of negative regulation of SMEK by MEK1 could be based on the existence of MEK1-SMEK complex that upon MEK1 activation and SUMOylation, recruits Ubiqutin Ligase MIP1. It is possible, that MIP1 ubiquitinates SMEK and targets this protein for proteasomal degradation. This could be a basis for negative regulation of SMEK by MEK1 signaling. Goals of the project:

• To prove that in Dictyostelium, MEK1, MIP and SMEK form multi-protein complex (using immune affinity purification and co-immunoprecipitation of tagged expressed versions of these proteins).

• To verify whether SMEK is ubiquitinated upon chemoattractant stimulation

• To check the dynamic composition of SMEK-MIP1 complexes over time after chemoattractant stimulation

• To compare steady-state levels of SMEK ubiquitination under the same conditions in cells either overexpressing MIP1 or possessing mip1 gene deletion

• In human cells (using suitable cell lines), validate RNF4 – hSMEK2 interaction

• To examine whether putative RNF4-hSMEK2 complex also contains MAP3K7, which was found in high throughput proteomics study of RNF4-interacting proteins.

• MAP3K7 is known to be ubiquitinated in response to cytokine activation and signaling. Does MAP3K7 ubiquitination require RNF4, as a component of StUbL? Is MAP3K7 also SUMOylated? Is SUMOylation a prerequisite for ubiquitination?

• To apply systems biology analysis of protein-protein interactions to verify existence of conserved multi-protein signaling complexes.


Reference names and contact information for application by Alex Sobko, Ph.D. Professor Valery Krizhanovsky,

Wolfson Building for Biological Research, Room 531a, Weizmann Institute of Science Rehovot, 76100, Israel


Phone: 972-*-*******

Stella Danet, PhD

Head of Laboratory of Aging and Cancer Research

Department of Molecular Biology, Ariel University, Ariel, 40700, Israel Phone:

Office: 972-*-*******

cell: 972-**-*******

Fax: 972-*-*******

Email:, Dr. Theresa C. White, Ph. D.

Carleton University, Ottawa, Ontario, Canada

Manager, Intellectual Property and Regulatory Affairs Agrisoma Biosciences, Inc., 200 Rue Montcalm, Suite 300, Gatineau, QC J8Y 3B5, Canada E-mail:


office: 613-***-**** x4024

Professor Bernard Attali,

Department of Physiology and Pharmacology, Sackler School of Medicine, Room 546 Tel Aviv University, Tel Aviv 69978, Israel


Phone: 972-*-***-****

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