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We are investigating the regulation of the MAP kinase pathway.  In particular, we have focused on how activation of this ubiquitous pathway can give rise to different outcomes in different cells.  As a first step in this work, we have used expression cloning to isolate proteins that interfere with an ERK MAP kinase signal to integrins.  Integrins are transmembrane heterodimers that mediate cell-cell and cell-extracellular matrix adhesion.  Activation of H-Ras, or its effector kinase, c-Raf-1, initiates a signaling pathway that impairs integrin ligand-binding.  This suppressor activity correlates with the activation of the ERK MAP kinase pathway. These changes in integrin activation may affect cell morphology, adhesion, and invasiveness. We used the H-Ras to integrin pathway as a tool to discover proteins that suppress ERK MAP kinase signaling.  One such protein is PEA-15. Several recent observations suggest that PEA-15 can affect ERK signaling to other proteins as well, including the transcription factor Elk1. 

PEA-15 is widely expressed throughout the body, with particularly high levels expressed in astrocytes.  The structure of PEA-15 is striking in that the N-terminal half of the protein consists of a conserved death effector domain (DED).  This domain, to date, is associated only with proteins involved in apoptosis. Indeed, PEA-15 prevents FADD-mediated apoptosis.  Moreover, the DED of PEA-15 is necessary, but not sufficient for PEA-15 blockage of the Ras to integrin signal.

We are using PEA-15 null mice and molecular and cell biological approaches to better understand the function of PEA-15 and its role in the development of cancers, including Breast cancer and Leukemia.

Background

MAP kinase pathways ERK pathwayTop of Page

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.

  ERK and Integrins

The ERK MAP kinase pathway

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 andThe ERK MAP kinase pathway restricts inactive ERK to the cytosol (Fukuda et al., 1997). The MEK and ERK complex dissociates when MEK is activated and phosphorylates ERK.  The ERK may then dimerize and this dimerization is apparently required for ERK to translocate into the nucleus by an active functions.

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 Top of PageExpression Cloning

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.

Expression cloning of candidate regulators of ERK MAP kinase

As described above, ERK has a variety of activities and not all those activities need occur in a given cell. Perhaps because Top of PagePEA-15 Structureof 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. While working with Dr. Mark Ginsberg, 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, we identified PEA-15 as a candidate novel regulator of the ERK MAP kinase pathway.

PEA-15  

Top of PagePEA-15 FunctionPEA-15 Structure

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).

PEA-15 Structure and Sequence

(A)

(B)

 

PEA-15 :   1 MAEYGTLLQDLTNNITLEDLEQLKSACKEDIPSEKSEEITTGSAWFSFLESHNKLDKDNL  60

FADD:      1 MDPFLVLLHSLSGSLSGNDLMELKFLCRERVSKRKLERVQSGLDLFTVLLEQNDLERGHT  60

Caspase 8: 2   DFSRNLYDIGEQLDSEDLASLKFLSLDYIPQRKQEPIKDALMLFQRLQEKRMLEESNL  59

                                                      *           *

PEA-15: 61 SYIEHIFEISRRPDLLTMVVDYRTRVLKISEEEELDTKLTRIPSAKKYKDIIRQPSEEEIIKLAPPPKKA 130

FADD:   61 GLLRELLASLRRHDLLQRLDDFEAGTATAAPPGEADLQV.................................99

Casp 8: 60 SFLKELLFRINRLDLLITYLNTR------KEEMERELQTPGRAQISAYRVMLYQISEE.............111

(A) Schematic showing general PEA-15 structure.  Important amino acid residues are indicated.  (B)  The full amino acid sequence of PEA-15 is shown aligned with two other FADD family members, FADD and Caspase 8.  Conserved amino acids are highlighted.  The phosphorylated serines are in red with asterices .

PEA-15 Function Top of Pageend of page

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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Web page created and maintained by Joe W. Ramos

Revised: February 2012

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