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| Background | |||
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MAP kinase pathways MAP Kinases play a central role in the growth, development and survival of all eukaryotic organisms. In mammalian cells, three major MAP kinase pathways have been characterized resulting in the activation of p42/44 known as extracellular regulated kinases (ERK), p38/SAPK2's and Jun kinases (JNK) (Lewis et al., 1998; Cobb, 1999). The activation of MAP kinases is regulated through control of initiation and propagation of cascades of kinases. In addition, MAP kinases are dephosphorylated and inactivated by multiple phosphatases, including dual specificity MAP kinase phosphatases (Waskiewicz and Cooper, 1995). Furthermore, the efficiency of MAP kinase activation is also affected by scaffolding proteins that assemble interacting components of MAP kinase cascades (Schaeffer and Weber, 1999; Schaeffer et al., 1998; Whitmarsh et al., 1998; Burack and Shaw, 2000; Karandikar and Cobb, 1999) . MAP kinases elicit their biological outputs by phosphorylating nuclear and cytoplasmic substrates.
The
canonical ERK MAP kinase cascade is stimulated upon the binding of
extracellular growth factors such as PDGF to their respective
transmembrane tyrosine kinase receptors.
The subsequent auto-phosphorylation of the cytoplasmic tails of
the receptor on tyrosine leads to the recruitment of grb2, which binds
the guanine exchange factor SOS. Recruitment of SOS to the membrane promotes its interaction with
the membrane localized H-Ras and results in GTP loading and activation
of H-Ras. This is followed
by the sequential recruitment and activation of Raf, MEK, and ERK. MEK binds and In yeast, MAP kinase pathways are directed in large part by scaffolding proteins that facilitate the interactions of specific constituents of a given pathway. More recently, scaffolding proteins were isolated in mammalian cells by yeast two-hybrid identification of proteins that bind specific members of the ERK MAP kinase pathway. These proteins include MP-1, which enhances MEK to ERK interactions and stimulates ERK signaling (Schaeffer et al., 1998) , and RKIP, which facilitates Raf to MEK binding, but inhibits ERK signaling (Yeung et al., 1999).
The ERK MAP kinase pathway suppresses
integrin affinity
Integrins are transmembrane heterodimers that mediate cell-cell and
cell-extracellular matrix adhesion
(Hynes,
1992) .
The affinity of some integrins for ligand is regulated by
“inside-out” cell signaling cascades (Hughes
and Pfaff, 1998;
Ginsberg, 1995).
Dynamic regulation of integrin affinity for ligand (activation) is
important in cell migration (Huttenlocher
et al., 1996), fibronectin matrix assembly (Wu
et al., 1995) ,
platelet aggregation in hemostasis and thrombosis (Martin-Bermudo
et al., 1998;
Ramos et al., 1996).
One pathway that regulates integrin activation is the ERK MAP kinase
pathway. Activation of the small GTP-binding protein H-Ras, or its
effector kinase, c-Raf-1, initiates an ERK-dependent signaling pathway
that suppresses integrin ligand-binding ("activation") (Hughes
et al., 1997).
Furthermore the Ras/ERK pathway can regulate cell shape and fibronectin
matrix assembly. Conversely, expression of a constitutively active form
of the small GTPase, R-Ras, can enhance integrin ligand binding (Zhang
et al., 1996). An
antibody that binds only active integrin, PAC1, is available and can be
used to monitor integrin activation state by flow cytometry .
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Expression cloning of candidate regulators of ERK
MAP kinase |
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As
described above, ERK has a variety of activities and not all those
activities need occur in a given cell.
Perhaps because 
of the potency of ERK signaling and its omnipresence, ERK is highly
regulated by a number of different kinds of proteins including
phosphatases, scaffolds, and kinases
(English
et al., 1999). To date,
these proteins have been identified as a result of their interaction
with constituents of the ERK pathway or the observation of a specific
protein's affect on the pathway.
I devised an expression cloning strategy that would potentially
identify proteins that negatively regulate a specific ERK signal: the
suppression of integrin activation (Ramos
et al., 1998) . I
recognized that any proteins identified by this screen could possibly
affect more than one or all ERK activities. The strategy consisted of transfecting a Chinese hamster
ovary (CHO) cell line with activated H-Ras and a cDNA expression
library. The cells were
sorted by flow cytometry and only cells that had active integrins in the
presence of high amounts of activated H-Ras were collected. The cDNAs
were isolated from these cells and amplified in bacteria.
These cDNAs were further enriched by re-transfection of cells in
batches and re-analysis by flow cytometry.
This process was repeated until a single cDNA was isolated that
would block the Ras/ERK signal to the integrins.
By this method, I identified PEA-15 as a candidate novel
regulator of the ERK MAP kinase pathway.
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| PEA-15 | |||
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PEA-15 (Phosphoprotein Enriched in Astrocytes) is a 15 KDa protein that was originally identified as a major astrocytic phosphoprotein (Estelles et al., 1996; Araujo et al., 1993). It maps to human chromosome 1q21.1 (Wolford et al., 2000; Hwang et al., 1997). This is a gene rich locus implicated in a number of diseases including acute myeloblastic leukemia and the skin disorder Ichthyosis vulgaris. The first 80 amino acids of PEA-15 correspond to the canonical Death Effector Domain (DED) sequence found in proteins that regulate apoptotic signaling pathways (Chinnaiyan et al., 1995; Boldin et al., 1996; Hu et al., 1997). The DED of PEA-15 can bind to the DEDs of both FADD and caspase 8 (Kitsberg et al., 1999; Condorelli et al., 1999). The remaining 51 amino acids contain a serine (S104) that is phosphorylated by PKC (Araujo et al., 1993) and a serine (S116) phosphorylated by calcium calmodulin kinase II
The 3’untranslated region (UTR) of PEA-15 has been independently cloned as a mammary transforming gene, called MAT1 (Bera et al., 1994) . PEA-15/MAT1 mRNA exists as 2.5 and 1.8 kb isoforms which differ by alternative splicing and polyadenylation (Tsukamoto et al., 2000; Estelles et al., 1996). The 3’ UTR contains several conserved regions that suggest the message may be tightly regulated (Estelles et al., 1996). Hence, an understanding of PEA-15 function will require a better knowledge of both mRNA and protein expression patterns. Thus far we know that PEA-15 is widely expressed in several tissues in addition to astrocytes, including lung, heart, spleen, kidney, thymus, and muscle while the protein has been detected in lung, eye, and fibroblasts (Estelles et al., 1996; Danziger et al., 1995).
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PEA-15 has so far been reported to affect three distinct cellular activities: ERK signaling, apoptosis, and glucose transport. I have previously reported that PEA-15 blocks the H-Ras to integrin signal by activating a pathway dependent on R-Ras (Ramos et al., 1998) . Furthermore, PEA-15 also activates the ERK MAP kinase pathway in a Ras-dependent manner. PEA-15 expression in CHO cells results in increased MEK and ERK activity and an increase in Ras GTP loading. Moreover, PEA-15 bypasses the anchorage-dependence of ERK activation (Ramos et al., 2000). These data strongly suggest that PEA-15 somehow interacts with the ERK MAP kinase pathway to regulate its activity.
PEA-15 was cloned as a protein with elevated expression in patients with
type II diabetes and was called PED-15 for Phosphoprotein enriched in
diabetes
(Condorelli
et al., 1998). They
found that overexpression of PEA-15 in L6 skeletal muscle cells
increases the number of Glut-1 transporters on the plasma membrane and
inhibits insulin-stimulated glucose transport and cell surface
recruitment of Glut-4. The
molecular mechanisms of PEA-15 function in this system has not been
characterized, but it is important to note in light of my data that
while the role of ERK in glucose transport is uncertain, based on our
data it is reasonable to predict that PEA-15 overexpression could affect
glucose stimulated ERK translocation .
PEA-15 contains a DED as stated above and has been reported to have a number of contradictory apoptotic functions depending on the cells examined. It is reported to protect astrocytes from TNFa-induced apoptosis (Kitsberg et al., 1999) , to inhibit artificially expressed Fas-mediated apoptosis in NIH 3T3 fibroblasts, to augment TNFa induced apoptosis in NIH 3T3 cells (Estelles et al., 1999), and to protect the transformed cells HeLa and MCF7 from both TNFa and Fas induced apoptosis . In some cases PEA-15 is shown to bind FADD or Caspase 8 DEDs (Condorelli et al., 1999; Kitsberg et al., 1999) and in one case it is shown not to bind FADD or Caspase 8 (Estelles et al., 1999). Moreover, PEA-15 phosphorylation on serines 104 and 116 is required for the reported affects on apoptosis in one of the reports (Estelles et al., 1999). It is not clear why there is a discrepancy in the reports with regard to PEA-15 interaction with FADD, but it may be due to differences in methodology or PEA-15 phosphorylation state.
Our knowledge of PEA-15 is currently very limited, as only a handful of papers have been published on it. Certain very basic questions remain to be answered: What is the molecular mechanism of PEA-15 action? What is the repertoire of proteins that PEA-15 can bind? When and where are PEA-15 mRNA and protein expressed? And what is the function of PEA-15 in the mouse? The answers to these questions will shed light on how PEA-15 has been implicated in Diabetes, oncogenic transformation, cell adhesion, apoptosis, and ERK signaling.
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We
are investigating the regulation of the
