Crosstalk of cJun with Sequence Specific Transcription Factors

c-Jun does not usually act alone, rather, it physically interacts with other transcription factors, allowing signal integration on promoter DNA for a combinatorial transcriptional regulation of gene expression. Interaction of this sort expands the diversity of c-Jun effects on gene regulation. For instance, it can bring c-Jun to a non-AP-1 site to regulate gene expression. c-Jun activates the monocyte-specific macrophage colony-stimulating factor (M-CSF) receptor promoter, which contains DNA binding sites for only PU.l, but not AP-1. c-Jun is recruited to the PU.l binding site in the M-CSF receptor promoter through binding to the Ets domain of PU.l for a transactivation effect (Behre et al, 1999). Binding to c-Jun does not always lead to transcription activation, as the c-Jun/MyoD complex suppresses the MyoD promoter and inhibits myogenesis (Bengal et al., 1992) and the c-Jun/Smad complex acts as a compressor for Smad3-mediated transactivation of TGF P responsive genes (Verrecchia et al., 2001; Dennler et al., 2000; Atfi et al., 1997). The tumor necrosis factor a (TNFa) and the human T-cell leukemia viral oncoprotein Tax agonize TGF p signaling, an effect likely resulting from the induction of c-Jun expression by TNFa and the increase of c-Jun N-terminal phosphorylation by Tax, both leading to c-Jun/Smad complex formation and abrogation of Smad binding to its target site for gene transcription (Arnulf et al, 2002).

Conversely, recruitment of Smad by c-Jun to the AP-1 binding sites synergistically activates AP-1-dependent promoters (Verrecchia et al., 2001). Apparently only some c-Jun binding proteins, including retinoblastoma (RB) protein and transcription factor CHOP, act as transcription co-activators on the AP-1 sites (Nead et al, 1998; Ubeda et al, 1999), while others act as co-repressors, such as MyoD and c-Jun dimerization protein 2 (JDP2) (Bengal et al, 1992; Heinrich et al., 2004). The c-Jun activation domain binding protein, JAB1, was originally identified as a c-Jun co-activator (Claret et al, 1996), as well as a component of the constitutive photomorphogenesis (COP9) signalosome. A fraction of JAB 1 is associated with the LFA-1 integrin at the plasma membrane. Induction of JAB1 nuclear translocation by the LFA-1 signal allows its association with c-Jun to potentiate AP-1-dependent promoter activity (Bianchi et al, 2000). JAB1 also brings to c-Jun the COP9 signalosome, consisting of protein kinase CK2 (CK2) and protein kinase D (PKD) that can phosphorylate c-Jun and block c-Jun-Ub conjugates and degradation (Uhle et al, 2003; Muller et al, 2000). Hence, proteins like JAB1 may act through multiple channels to activate c-Jun-mediated transcription.

Gene promoters usually contain binding sequences for several transcription factors, such that promoter activity is determined by these factors acting in synergy. This appears to be the case for the tissue inhibitor of metalloproteinases-1 (Timp-1) promoter, containing adjacent Ets and AP-1 binding sites. The optimal promoter activity requires the binding of a trimolecular complex, consisting c-Jun, Ets and Fos, which is particularly efficient in DNA binding and synergistically activating transcription (Bassuk and Leiden, 1995; Logan et al, 1996). The optimal activation of the osteopontin (Opn) promoter derives from the interaction and cooperation of beta-catenin/Lef-1, Ets, and AP-1 transcription factors (El Tanani et al, 2004). Likewise, interactions of c-Jun and NFATp or c-Jun and Stat3 are responsible for the maximal transcriptional activity of specific promoters containing closely adjacent binding sequences for respective transcription factors (Zhang et al, 1999a; Alroy et al, 1995). Hence, association with other transcription factors provides ample explanation for the diversity, the intensity and the specificity exerted by c-Jun that sometimes activates and others suppresses gene transcription.

Signaling Mechanisms Involved in c-Jun Phosphorylation

Transmission of the extracellular signals through the cytoplasm is mediated by cascades of protein kinases, leading to changes of the transcription factor phosphorylation state and modulation of transcription activity (Karin, 1991; Bohmann, 1990). Glycogen synthase kinase 3 (GSK-3) and CKII can phosphorylate c-Jun at its C-termini, keeping c-Jun in a non DNA-binding state (Boyle et al, 1991), while extracellular signal regulated kinase (ERK)-mediated activation of p70 S6 kinase phosphorylates GSK-3 at serine-21 and inactivates it (Sutherland et al, 1994). It is therefore possible that ERK activation leads to c-Jun DNA binding activity through p70 S6 kinase-mediated GSK-3 inactivation and c-Jun C-terminal dephosphorylation (Fig.13.1). A connection between ERK and c-Jun has been suggested by mutation studies in fission yeast, of which homologs of mammalian ERK and Jun act in concert to control yeast cell elongation immediately after division (Toda et al, 1991). The ERK, however, does not seem to be involved in modulating the c-Jun N-terminal phosphorylation, which must be catalyzed by other protein kinases (Westwick et al, 1994).

The c-Jun ammo-terminal kinases (JNKs) have been identified based on their activation by UV and oncoproteins. Like the ERKs, the JNKs are proline-directed kinases with optimal sequence Pro-Xaa-Ser/ Thr-Pro for phosphorylation; while unlike the ERKs, the JNKs have distinct substrate specificity, being unable to phosphorylate pp90rsk but more efficient in phosphorylating the c-Jun transactivation domain (Fig.13.1). JNK phosphorylates c-Jun at serines 63 and 73 and phosphorylation requires binding of JNK to a specific region within the c-Jun transactivation domain (Hibi et al., 1993; Derijard et al., 1994; Kyriakis et al., 1994). This feature explains why v-Jun, having completely conserved Ser-63 to Ser-73, but lacking the JNK binding domain (amino acids 34-60), is resistant to TPA-induced N-terminal phosphorylation (Adler et al., 1992).

The MAP Kinase Cascade

The JNKs and the ERKs, together with the later discovered p38s, constitute three separate groups of Mitogen-Activated Protein Kinases (MAPKs), which are themselves activated through concomitant phosphorylation on tyrosine and threonine residues in the Thr-Xxx-Tyr motif (Fig. 13.2). For each MAPK group, the phosphorylation is catalyzed by the designated MAP kinase kinases (MAPKKs), a novel class of dual specific protein kinases (Mordret, 1993). The connection between MAPKK to the MAPK is fairly specific, as the MEK4 and MEK7 activate the JNKs, the MEK1 and MEK2 activate the ERKs, and the MEK3 and MEK6 activate the p38s (Johnson and Lapadat, 2002). The activities of the MAPKKs are turned on by serine/threonine phosphorylation catalyzed by their immediate upstream kinases, the MAPKK kinases (MAPKKKs) (Davis, 1994). A rapidly increasing number of protein kinases are categorized as MAPKKKs, including Rafs, MEK kinase 1-4 (MEKKs), germinal center kinase (GCK), mixed lineage kinases (MLK), apoptosis-stimulated kinase 1(ASK1), tumor progression locus 2 (TPL2), and TGF-beta-activated kinase (TAK). Having rather distinct regulatory motifs for connection to unique input signals, but relatively conserved kinase domains for activating the

MAPKK-MAPK cascade, the MAPKKKs appear to be exactly what is needed for conferring numerous signals to MAPK activation (Schlesinger et al., 1998). The complex role for MAPKKK in cell signaling is best illustrated by studies on the regulation and function of MEKKl (Xia and Karin, 2004).

MEKK1 was originally identified as a mammalian homolog of the yeast MEK kinases, Byr2 and Stell, involved in pheromone-induced mating (Lange-Carter et al., 1993). Although initially considered as an upstream activator of ERK, it soon becomes clear that MEKKl preferentially activate the JNK pathway, by interacting with and phosphorylating the JNK activator, MEK4 (Minden et al., 1994; Yan et al., 1994; Lin et al., 1995; Xia et al., 1998). MEKKl is in turn activated by various upstream signals, and intriguingly, each signal appears to be coupled to MEKKl by a distinct mechanism. The diversity of MEKKl in cell signaling is attributed, in part, to its N-terminal regulatory domain, which can interact with various upstream regulators to establish a connection (Fig. 13.3). Binding to the SH3 domain of Grb2 through this region allows the recruitment of the MEKK1/Grb2 complex by the She proteins to the activated EGF receptor and a transient plasma membrane localization of MEKKl (Pomerance et al., 1998). The membrane localized MEKKl undergoes conformational changes to initiate autoactivation, by which MEKKl phosphorylates its own threonine residues between the kinase subdomains VII and VIII (Deak and Templeton, 1997; Siow et al., 1997). The N-terminal domain of MEKKl also offers binding sites for RhoA, JNK and alpha-actinin, while its C-terminal kinase domain binds MEK4 (Gallagher et al., 2004; Christerson et al., 1999; Xu et al., 1996; Xia et al., 1998).

Cytoplasm MAPKKK

Physiological and environmental factors

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