CURRENT RESEARCH AND PROJECTS IN THE LABORATORY
Cell Signalling group
Signalling via ribosomal S6 kinases
The emphasis of this project is on elucidating the role of ribosomal S6 kinase
(see below), a downstream effector of PI3-K, via the identification and functional
characterization of S6K-binding partners. The technique of yeast two-hybrid (Y2H)
screening has been established in the laboratory and extensively
used for screening mouse embryo library with various S6K bait constructs.
Using this approach, we have identified a novel protein, termed CoA synthase, as a
binding partner for S6K. Furthermore, we showed by biochemical and mutational
studies that CoA synthase possesses two enzymatic activities, which mediates
the last steps in CoA biosynthesis. These findings provide an interesting link
between S6K signaling pathway and cellular metabolism. Further studies on the
regulation of CoA synthase and the role of S6K in this process are currently
in progress. In addition, the use of activated versions of S6Kα and β
as baits allowed us to identify 3 additional binding partners, which are specific
for activated forms of S6K. Functional characterization of these interactions
in mammalian cells is currently under examination.
PTEN - antagonist of PI3K signaling pathway
PTEN (phosphatase and tensin homologue deleted on chromosome ten) also referred to as MMAC (mutated in multiple advanced cancers)
was discovered as a tumor suppressor gene, which is frequently mutated in most human cancers.
Numerous studies demonstrated that PTEN possesses phospholipid phosphatase activity which acts
on phosphatidylinositol 3,4,5-triphosphate and, therefore, down-regulates PI3-kinase signaling pathway.
In addition, it displays tyrosine phosphatase activity that has been implicated in controlling integrin-mediated
adhesion; apoptosis and cell cycling through various downstream targets, including FAK, Src and PKB/Akt.
The main goal of this project is to identify novel regulator/s of PTEN enzymatic activity. Identification
of novel interactiors of PTEN may provide new insight into the biology of normal and malignant cells.
A panel of bait vectors, containing C-terminal domain of PTEN, full-length wild type, phosphatase-dead and activated forms has been constructed. The expression of all constructs in yeast was confirmed by transformation
and immune blotting with specific anti-Lex A antibodies. Nuclear translocation and autoactivation assays provided
the information about the suitability of generated constructs for library screening. Human Colon Cancer, Mouse
embryo and HeLa cDNA libraries have been screened with full length and C-terminal PTEN baits. These screens allowed
us to isolate 110 clones with the C-terminal bait and 11 with the full length PTEN. Currently, the analysis of all clones by mating assay in yeast is in progress. Simultaneously, isolated clones were rescued from yeast, re-transformed into KC8 bacterial cells and sequenced.
We hope that successful outcome of this study would allow us to identify novel regulatory mechanisms in PI3-K signaling pathway.
Cancer Immunology group
The development of specific immunotherapies of human malignancies is based
on explicit recognition by the immune system of tumor-specific or tumor-associated
antigens, which are either specifically expressed or overexpressed in cancer
when compared with normal cell or tissues. Therefore, the search for tumor-associated
antigens has been the subject of intense research for many years. Recent advances
in molecular biology and immunology have created the platform for the identification
of novel tumor-associated antigens by various approaches, including T-cell epitope
cloning, SEREX methodology, differential display and SAGE.
The main objective of this project is to identify tumor-associated antigens
from melanoma, thyroid and colon cancer by applying SEREX methodology (Serological
identification of antigens by recombinant expression cloning). We have generated
a panel of cDNA expression libraries from tumor samples and screened them with
affinity purified sera from corresponding patients. In the course of this study
we have isolated more than 100 serum positive clones, which have been identified
by DNA sequencing. These clones have been submitted to a SEREX database,
established by the LICR. We are currently analyzing isolated clones by heterologous
screening with the aim to select the candidates for further and more detailed
analyses. Knowledge of the nature and expression profiles of novel cancer-specific
antigens, identified in this study, will form the basis for future development
of new diagnostic approaches and immunotherapies of cancer. This project is part
of the SEREX Program, which is coordinated by the Ludwig Institute for Cancer
Research (LICR).
Protein Biochemistry group
Functional analysis of A33 antigen (in collaboration with LICR, New York Branch)
A33 antigen is a transmembrane protein, which belongs to the subfamily within the immunoglobulin superfamily,
which includes CTX/ChT1, CTM/CTH, and CAR. A33 is expressed in normal gastrointestinal epithelium and in 95%
of human colon cancer. Taking this into account and the absence of A33 expression in most other human tissues,
this antigen is the focus of several clinical studies in patients with colon cancer.
The function of A33 antigen is not known. The aim of this collaborative project is to identify A33-binding
partners and to characterize functional importance of these interactions. The techniques of yeast two-hybrid
screening and affinity co-purification followed by mass spectrometry have been employed in the this study.
Using these approaches, we have identified 2 binding partners (one by Y2H screen and one by affinity co-purification).
These interactions have been confirmed in mammalian cells by co-immunoprecipitation assay, followed by Western blotting.
One of the identified binding partners links A33 to actin cytoskeleton.
Monoclonal antibody group
Additional studies are focused on generation of specific tools for functional analysis of S6K.
We have recently developed a panel of monoclonal antibodies specific for both S6Kα and S6Kβ isoforms.
The availability of these antibodies allowed us to study subcellular localization and the pattern
of expression of S6Ks in normal and cancer cell lines and tissues. Moreover, we are going to use them
as an affinity purification matrix for isolation of S6K-binding partners. We have recently generated a
panel of monoclonal antibodies specific towards CoA synthase.
The development of monoclonal antibodies against tumor suppressors PTEN, TSC1/2 complex is currently in progress.
Ribosomal S6 kinase
Ribosomal S6 kinase (S6K) belongs to the AGC family of Ser/Thr protein kinases,
which includes the PKCs, PKBs, SGKs and p90 RSK. There are two forms of S6K, S6Kα and S6Kβ,
which have cytoplasmic and nuclear variants derived from alternative splicing.
The activity of S6K is regulated by phoshorylation / dephosphorylation events in cellular
responses to various extracellular stimuli. The mechanism of activation of S6K is a multi-step
process which is achieved by coordinated phosphorylations at three regions: the C-terminal
autoinhibitory domain, by a Ser-Pro directed kinases; the activation loop in the kinase domain by PDK1;
and the conserved hydrophobic site in the kinase-extension domain.
No direct, highly specific S6K inhibitor has yet been identified.
Under these circumstances, the use of two indirect inhibitors, namely wortmannin (PI3-K inhibitor)
and rapamycin (Tor kinase inhibitor), has been instrumental in dissecting signaling events
involved in the regulation of both forms of S6K. Studies from numerous laboratories demonstrated
that signals from PI3-K and Tor kinase pathways are crucial for full activation of S6Kα.
A number of upstream effectors of S6K have been identified, including PDK1, PKB/Akt,
atypical isoforms of PKC and the small GTP-binding proteins Rac and CDC42.
Ribosomal protein S6 is the main physiological substrate of S6K.
The phosphorylation of S6 protein was shown to closely correlate with the initiation
of protein synthesis induced by various extracellular stimuli. The transcriptional activator CREM,
elongation factor 2 kinase and the regulator of apoptosis, Bad 1 were also shown to be phosphorylated by S6Kα
in vitro and in vivo. However, the physiological relevance of these phosphorylations
requires further investigation, since other protein kinases can phosphorylate these molecules at identical sites.
Taking into account that S6Kα was identified more than a decade ago, but S6Kβ only recently,
most functional studies have involved the p70/p85 isoforms of S6Kα. S6Kα
was found to be involved in translational up-regulation of a specific pool of mRNAs,
containing an oligopyrimidine tract at the 5` transcriptional start site (5` TOP mRNAs).
As 5` TOP mRNAs encode components of the translational machinery, such as ribosomal proteins,
elongation factors and poly(A) binding proteins, it has been proposed that S6K can regulate ribosome biogenesis,
through phosphorylation of the S6 ribosomal protein. Microinjection studies with neutralizing antibodies
against S6Ka demonstrated its importance in mediating G1/S transition of the cell cycle.
(Lane et al., 1993; Reinhard et al., 1994.)
Knock-out of the S6Ka gene in mice and Drosophila indicated that the kinase is a key player
in the regulation of cell size, growth and glucose homeostasis. Mice deficient in the S6Kα gene
exhibit a significant reduction in body size during embryonic development.
This effect is largely corrected by adulthood possibly via compensatory function of S6Kβ,
whose expression is elevated in mice deficient for S6Kα. Detailed analysis of glucose homeostasis
in S6K deficient mice revealed a selective decrease in pancreatic β-cell size, which is accompanied
by hypoinsulinaemia and glucose intolerance.
The observed phenotype resembles some aspects of type 2 diabetes mellitus.
Deletion of S6K in Drosophila, which possess only one form of the kinase,
leads to a higher incidence of embryonic lethality, an extreme delay in development
and a severe reduction in body size. Further genetic and biochemical studies in Drosophila
indicated that regulation of cell growth via dS6K requires the input from dPDK1, but is dPKB/dPI3-K independent.
As mentioned above, S6Kα and S6Kβ are each represented by two splicing variants
with distinct subcellular distribution. The 23- and 13-amino acid extensions at the N-termini
of S6KαI and S6KβI contain nuclear localisation signals that target these isoforms
constitutively to the nucleus. The cytoplasmic form of S6Kα (S6KαII) or p70 S6K
is predominantly cytosolic, but accumulates in the nucleus when cells are treated with Leptomycin B.
The presence of a functional nuclear localisation signal at the C-terminus of S6Kβ,
which is common for both splicing variants, has been recently reported. (Koh et al., 1999.)
The nuclear functions of S6Ks are not known. It has been proposed that in the nucleus,
S6Ks may be responsible for the phosphorylation of the free, chromatin-bound form of S6 protein
and the initiation of ribosome biogenesis.
|
