Program
One:
Signal transduction and gene regulation mechanisms are being studied by analyzing
Mitogen-Activated Protein Kinase (MAP kinase) control of the regulatory transcription
factor Activator Protein-1 (AP-1). MAP kinases are signal transduction intermediates
in responses to endrogenous and environmental stimuli for diverse intracellular
functions including growth regulation, carcinogenesis, and development. The
AP-1 transcription factor controls expression of effector genes which execute
physiologic responses to numerous extracellular stimuli such as growth factors
and tumor promoters. MAPKs are upstream regulators of AP-1. The mechanisms
of AP-1 regulation by MAPKs in vivo is an area of intensive study and interest.
To date there has been controversy as to whether MAP kinase regulation of
AP-1 involves direct MAPK phosphorylation of AP-1 components or is indirect
via signalling intermediates between MAPK and AP-1. By immunoprecipitation
and comparative two dimensional gel electrophoretic analyses of proteins associated
with AP-1 we have identified a family of seven MAPK related proteins (MAPKRPS),
including ERK-2, which bind AP-1 in vivo. Identification of these complexes
was achieved by isolating them by immunoprecipitation with antibodies against
AP-1 (Jun and Fos family) proteins. Proteins with the same molecular weights
as MAP kinase-related proteins are co-immunoprecipitated from whole cell extracts
using a variet
y
of antibodies directed against AP-1 proteins (see figure 1). Comparative
two-dimensional electrophoretic analyses of MAPKRPs to the co-immunoprecipitated
proteins that co-migrate with MAPKRSs reveals that the proteins associated
with AP-1 are in fact MAPKRPs. The MAPKRPs bind to multiple AP-1 dimers composed
of a variety of Jun and Fos family AP-1 partners. AP-1 regulation by MAPKs
thus appears to occur at least in part through direct protein-protein interaction,
in addition to kinase-substrate interaction. We are cloning genes encoding
new MAP kinase-related proteins. Kinase activity of MAPKRPs is being assessed
by in vitro assays with AP-1 components as substrates. The functional significance
of the multiplicity of stable MAPKRP-AP-1 complexes is under investigation.
The roles of MAPKRP-AP-1 complexes in susceptibility to neoplastic transformation
are also being studied. This program is likely to lead to cloning of one or
more new MAPK genes and shed light on mechanisms of MAPK and AP-1 signal transduction.
Figure 1. AP-1 antibodies co-immunoprecipitate antigens with the same molecular weights as MAP kinase-related proteins. Lane 1, non-immune rabbit serum; lane 2, MAPK antibody a -ERK1-III (Upstate Biotechnologies against residues 63-98 of rat ERK-1); lane 3, a -c-Jun Ab-II (against residues 255-335 of human c-Jun); lane 4, a -Fos antibody (Upstate Biotechnologies, Inc., against residues 75-155, avian v-Fos).
Program
Two:
This program is a new program whose purpose is to identify
determinants of susceptibility
to neoplastic transformation. We are particularly interested in identifying
determinants of susceptibility to instability within the genome since we postulate
that these are underlying factors which make cells susceptible to cancer.
We are creating a model system to address questions, employing genetic cellular
variants which harbor differential susceptibility to neoplastic transformation
induced by phorbol ester tumor promoters. Our goal is to clone genes which
specify this susceptibility.
Figure 2. Proteins co-precipitated with Jun and Fos proteins are identical to MAP Kinase-Related Proteins. A. Two-dimensional analysis of proteins immunoprecipitated by a -ERK1-III. MAPKRP protein spots A (p40), B(p38), C(p36), D(p33), p49, p65 and p96 are indicated. Spots E (39 kDa), X (42 kDa), and Y (35 KdA) are also reproducibly detected. B, a -ERK1-III plus peptide antigen symbols show orientation of poles for isoelectric focussing in the first dimension. Mr represents molecular weight standards in the second dimension