Research Field

Cell growth, cell surface receptor signaling, signal transduction mechanisms, recognition domains and drug design.

Research Activity

Ras and heterotrimeric GTP binding proteins are major components of cell growth signaling pathways that are triggered by ligand-activated tyrosine kinase or seven-transmembrane-domain receptors. Both Ras proteins and the g subunits of heterotrimeric G pro teins contain a carboxyl-terminal S-prenylcysteine (SPC) which is absolutely required for their membrane anchorage and biological activities as mediators in signal transduction. Independent studies performed by us and by others revealed that prenylcystein e mimetics specifically interfere with the interactions of activated seven transmembrane-domain receptors and heterotrimeric G proteins as well as with the membrane anchorage and functions of Ras proteins.

One of our major goals is the identification of a presumably distinctive Ras SPC-docking site and the characterization of its role and significance in Ras-mediated cell-growth signaling. We have recently demonstrated for the first time the selective dislo dging of Ras from intact Ras-transformed cell membranes by the unique SPC mimetic, S-farnesylthiosalysilic acid (FTS). Consequently, the cell’s transformed-phenotype is reversed, and cell- and tumor-growth in nude mice are inhibited. Structure activity re lationships among FTS analogues, and selective inhibition of growth signals upstream, but not downstream, of Ras demonstrate FTS specificity and selectivity. These results provide a strong support for the existence of distinctive, yet undetected, SPC anch orage sites for Ras in cell membranes, as well as new experimental tools for the identification of such sites, and a novel concept of competing Ras from its sites as a potential method for cancer chemotherapy. We attempt now to demonstrate that Ras or het erologous proteins carrying the Ras docking sequence can be dislodged from the plasma membrane by specific SPC mimetics even without an effect on Ras processing, to determine the consequences of this effect on the stability of the dislodged Ras and on the activity of down-stream growth signaling components and on cell growth and attempt to label, tag, identify and isolate the Ras docking site. FTS and other SPC mimetics interfering with Ras functions, are used to affect Ras-mediated activation of c-Raf1 k inase and mitogen-activated protein kinase (MAPK), in Rat1 fibroblasts stably expressing activated Raf, Ras, tyrosine kinase receptors, partially processed Ras and Raf targeted to the membrane by a Ras targeting signal sequence (Raf-CTR). The nature of th e FTS-elicited dislodging of various ras mutants and of Raf-CTR from cell membranes, and the fate of the dislodged proteins are studied by combined immunoprecipitation/immunoblot assays. The relationships between Ras-dislodging and the structure of SPC-m imetics and their effects on Raf-MAPK is now being determined. These experiments, together with radioligand binding assays ([3H]FTS) and affinity labeling ([3H]FTS-mustard) combined with electrophoretic and chromatographic methods should lead to identific ation of SPC sites for Ras.

A second major goal in our studies is the identification of SPC sites for Gbg. We perform functional analysis of the effects of SPC mimetics on the interaction of G protein bg subunits with known regulatory proteins (e.g. guanine nucleotide exchange facto rs, GTPase-regulating proteins, downstream targets like components of the pathway for Ras proteins). In these studies we use both intact cells, that express Gi/Go proteins and seven-transmembrane-domains-receptors (muscarinic, LPA, thrombine, receptors), and cell free systems where G proteins, phospholipase-C(, hSosl and K-RasB purified from baculovirus-infected insect cells are reconstituted. Our studies are expected to provide new information on the nature and on the structural properties of SPC domains and could help in the design of new strategies to alleviate certain types of human cancers and to block undesired cell proliferation in many other types of human diseases.

1. Klein, Z., Ben-Baruch, G., Marciano, D., Solomon, R., Altarat, M. & Kloog, Y. (1994) Characterization of the prenylated protein methyltransferase in human endometrial carcinoma. Biochem. Biophys. Acta 1226, 330-336.
2. Marciano, D., Ben-Baruch, G., Marom, M., Egozi, Y., Haklai, R. & Kloog, Y. (1995) Farnesyl derivatives of rigid carboxylic acids-inhibitors of ras-dependent cell growth. J. Med. Chem. 38, 1267-1272.
3. Marom, M., Haklai, R., Ben-Baruch, G., Marciano, D., Egozi, Y. & Kloog, Y. (1995) Selective inhibition of ras-dependent cell growth by farnesyl thiosalicylic acid. J. Biol. Chem. 22263-22270.

1. Supplementary data:

   You can find our articles supplementary data in here!

   

The Kloogs have fun ALL times

The Main Kloog

Yoel Kloog

Laboratory administrator

Haklai Roni

  and 

Post-Doc Students

Elad-Sfadia Galit

PhD. Students

Yaari-Stark Shira

My research focused on Ras and Myc cooperation, which contributes to the malignant phenotype of human cancers with chronically-activated Ras and an amplified Myc gene. To address this question, I used the Ras inhibitor S-farnesylthiosalicylic acid (FTS, salirasib®).

I showed that Active Ras is needed to block MycN degradation, thus promoting cooperative Ras- and MycN-dependent cell-cycle progression in LAN-1 human neuroblastoma cells.

In the search for a common response of amplified – Myc cell lines (LAN-1, NCIH929, and K562) to FTS treatment, I used global gene analysis of the three cell lines, combined with functional analysis and promoter sequence analysis. Gene expression analysis enabled me to decipher the common and most prominent patterns of gene expression response of those cells treated with FTS and indicated a decrease in the transcription factors E2F and NF-Y, which are critical for cell cycle progression. I also identified a unique common response to FTS, namely, enrichment of genes that were increased and are controlled by the transcription factor Nrf2. These results suggest that FTS not only causes cell-cycle arrest but also appears to induce a stress response in amplified Myc gene cells. For this purpose I utilized RT-PCR, siRNA, shRNA and combined inhibitors approaches.

1.  Yaari, S., Jacob-Hirsch, J., Amariglio, N., Haklai, R., Rechavi, G., and Kloog, Y.   Disruption of cooperation between Ras and MycN in human neuroblastoma cells promotes growth arrest. Clin. Cancer Res. 2005; 11: 4321-4330.

2. Blum, R., Elkon R., Yaari, S., Zundelevich, A., Jacob-Hirsch, J., Rechavi, G., Shamir, R., and Kloog, Y. Gene expression signature of human cancer cell lines treated with the ras inhibitor salirasib (S-farnesylthiosalicylic acid). Cancer Res. 2007; 67(7):3320-8.

Barkan Batya

Mor Adi

Rechavi Oded

Studies a novel phenomenon we identified in the lab: cell-to-cell transfer of intracellular signaling proteins. To characterize such transfer mechanisms I utilize advance mass-spectrometry tools, genomic technologies (SOLEXA), FACS and microscopy. Recently I have been working on small RNAs and Prion proteins as well.

Levy Ran

Rak Roni

I'm currently at the first year of my PhD studies in the Dean's list program for outstanding students of the Tel Aviv University. My research is focused mostly on the study of recently discovered members of the Ras and Rho family proteins, Eras (embryonic stem-cell expressed Ras) and WRCH1 (Ras homolog in the brain). I am particularly interested in their tumor progressing functions as they appear to be governed by different subcellular localizations and post-translational modifications.

MSc. Students

Taragan Anat

Feder Orna

Makovski Victoria

Aizman Elizabeta

Past PhD. Students

  Rotblat Barak                     Blum Roy                     Shalom-Feuerstein Ruby

             Peretzman Adi                                        Starinsky Sigal                                             Goldberg Liat
         

Past MSc. Students

1. Belanis Liron                    2. Nakdimon Itay                                 3. Zilberberg Roi

Some more pictures:

Sample of our studies:

“Ras micro/macro localization is coordinated by its lipids, C'-terminal sequences and G-domain ”/Rotblat Barak

My studies focused on Ras where my broad goal was to understand how Ras signaling is coordinated.  To this end I decided first to learn about the contribution of the H-Ras C-terminal domain to the lateral organization of the protein in the plasma membrane. I used GFP-fused mutants of this domain and performed biophysical measurements in live cells.  In these experiments I employed the FRAP (fluorescence recovery after photobleaching) method and found that three separable domains regulate GTP-dependent association of Ras with the plasma membrane. This work was published (Rotblat et al., Mol Cell Biol. 2004 Aug;24(15):6799-810.). In a follow-up study, we found that individual palmitoyl residues serve distinct roles in H-Ras trafficking, microlocalization and signaling (Roy et. al., Mol Cell Biol. 2005 Aug;25(15):6722-33.). These studies were in collaboration with the JF Hancock lab, Brisbane Australia. 

 

Rotblat et al., Ras and its signals traverse the cell on randomly moving nanoparticles, Cancer Res.See also movies section below):

Movies section:

Rasosomes diffuse out of digitonin treated cells
A BHK cell expressing GFP-H-Ras was treated with digitonin for 10 min and imaged live under epi- fluorescence at 100 ms/frame.

Classic vesicular transport is ATP dependent

BHK cell expressing YFP-NPY that labels exocitic vesicles was imaged live under epi-fluorescence at 200 ms/frame. Before ATP depletion (right) after ATP depletion (left).

Rasosomes diffuse near or at the plasma membrane (TIRF microscopy)

A BHK cell expressing GFP-H-Ras was treated with digitonin for 10 min and imaged live under TIRF microscopy at 100 ms/frame.

 

Rasosomes diffuse in the cytoplasm

A BHK cell expressing GFP-H-Ras was treated with digitonin for 10 min and imaged live under epi- fluorescence at 100 ms/frame.

 

The scene of crime

1. SIGNALING PATHWAYS CONTROLLING CELL DIFFERENTIATION, CELLGROWTH AND APOPTOSIS