Multimeric Complexing Of Ers With Signaling Proteins

Critical and as yet unanswered questions concern the identity of the pathways that may mediate estrogen-induced activation of the ERKs in the brain and by which neurotrophins elicit tyrosine phosphorylation of the unliganded neural ER. The MAP kinase cascade is an important pathway for NGF-induced differentiation in PC12 cells. Although it is presumed that neurotrophin signaling pathways in the brain are likely to be similar to those of PC12 cells, the actual cascades used have never really been characterized in the CNS. In PC12 cells, signal transduction through the MAP kinase cascade is dependent on activation of Ras, which binds directly to the Raf family of protein kinases to mediate their translocation to the membrane, essential for Raf activation. The Raf kinases (b-Raf in PC12 cells; c-Raf (Raf-1) in sympathetic neurons; ?? b- and/or c-Raf in the CNS) initiate the MAP kinase (MAPK) cascade by phosphorylating MEK (MAPK kinase) which in turn phosphorylates and activates MAPK or ERK. The b-Raf is the only one of the Raf family members to become phosphorylated in response to NGF exposure of PC12 cells (112). In PC12 cells, b-Raf has been found to be present as a component of a putative high molecular weight (>300 kDa) complex, consisting of at least b-Raf and heat shock protein (hsp) 90, whose assembly is constitutive and independent of NGF activation (113). Hsp90 is highly abundant in unstressed eukaryotic cells and represents 1-2% of the cytosolic protein (114).

We have recently found evidence that this putative complex is present in the CNS (cerebral cortex) as well (49,116). Because the unliganded ER is believed to be kept inactive in the cytosol and from forming homodimers by becoming complexed with the hsp90-based chaperone system (28,114), we started investigating whether the putative b-Raf/hsp90 complex may also include the unliganded ER. Like the ER, hsp90 becomes tyrosine-phosphorylated by estrogen. In addition, because hsp90 has also been reported to form stable complexes with pp60src (28,114) as well as the ER, we hypothesized that src, a nonreceptor protein tyrosine kinase, would also be a good candidate, because estrogen has been reported to elicit tyrosine phosphorylation of src within 10 s (63). In fact, Migliaccio et al. (65) have suggested that estrogen-induced tyrosine phosphorylation of the ER is a consequence of the very rapid phosphorylation of src. We further questioned whether, in the CNS, other protein kinases downstream of Raf in the MAP kinase cascade, e.g., MEK and ERK, might also be associated with both hsp90 and the ER in a multimeric complex or even in more than one complex, depending on the ER type.

Preliminary immunoprecipitation experiments in explants of the cerebral cortex and a PC12 cell variant, PC12-E2 (116), which expresses high levels of ERs (M. Singh and

D. Toran-Allerand, unpublished observations) show evidence by Western analysis of co-immunoprecipitation of b-Raf, src, and hsp90 with the ER and of MEK with src (49,115). Surprisingly, co-precipitation of hsp90 with ERK and of the ER with ERK has also been documented in the cortical explants (117). The sum of these findings implies that these signaling proteins may also be components of the putative complex or complexes. One might speculate, as illustrated in Fig. 6, that, following neuronal exposure to

E, dissociation and conformational changes within the complex(es), resulting from tyrosine phosphorylation of the ER, hsp90, and src, may lead to direct phosphory-lation and activation of MEK and thence ERK. Alternatively, estrogen exposure could even lead to phosphorylation and activation of ERK directly via a putative ERK/ ER/hsp90 complex.

Conversely, because activation of trkA following NGF exposure also elicits tyrosine phosphorylation and activation of MEK via Ras/Raf activation, this hypothesis also suggests a reciprocal signaling pathway by which the unliganded ER, bound by hsp90 to the rest of the putative multimeric complex(es), could become tyrosine phosphorylated by the neurotrophins. Thus, regardless of the ligand, dissociation of the ER from these putative complexes, consequent to tyrosine phosphorylation within the complex, would be not only ER activation but activation of ERK as well, followed by nuclear translocation of both the ER and ERK, and gene regulation.

Despite the issues of ER subtypes and affinities raised earlier, that the ER may be complexed directly with MEK, the kinase immediately upstream of ERK, or complexed

Fig. 6. Sites of potential cross-coupling of the estrogen and neurotrophin receptor systems. Estrogen and the neurotrophin-induced activation of the putative multimeric b-Rafcomplex by tyrosine phosphorylation, which would be accompanied by changes in the configuration of its hsp90/src/ ER components, may phosphorylate MEK and lead to rapid activation of MAPK/ERK. Alternatively, estrogen-induced activation of the putative ER/hsp90/ERK complex may phosphorylate ERK directly. These changes would be followed in both instances by nuclear translocation of both ERK and the now-activated ER, resulting in activation of immediate and late response genes and transcription. On the other hand, activation of the trk receptors and of the downstream putative b-Raf complex by NGF and the other neurotrophins could lead, conversely, to tyrosine phosphorylation of the unliganded ER.

Fig. 6. Sites of potential cross-coupling of the estrogen and neurotrophin receptor systems. Estrogen and the neurotrophin-induced activation of the putative multimeric b-Rafcomplex by tyrosine phosphorylation, which would be accompanied by changes in the configuration of its hsp90/src/ ER components, may phosphorylate MEK and lead to rapid activation of MAPK/ERK. Alternatively, estrogen-induced activation of the putative ER/hsp90/ERK complex may phosphorylate ERK directly. These changes would be followed in both instances by nuclear translocation of both ERK and the now-activated ER, resulting in activation of immediate and late response genes and transcription. On the other hand, activation of the trk receptors and of the downstream putative b-Raf complex by NGF and the other neurotrophins could lead, conversely, to tyrosine phosphorylation of the unliganded ER.

directly with ERK itself has strong implications for estrogen activation of ERK. Direct activation of these putative complexes by both estrogen and the neurotrophins would also have significant implications for the rapidity and specificity of their activation of immediate-early genes and nuclear transcription factors. Segnitz and Gehring (28) have suggested that the nonactivated ER of human MCF-7 mammary carcinoma cells has a heterotetrameric structure consisting of one receptor polypeptide, two hsp90 molecules, and one p59 subunit, for which the molecular mass adds up to approximately 300 kDa. No other proteins, including the heat-shock protein hsp70 and the 40-kDa cyclophilin, were reportedly detected as components of this highly purified cross-linked ER complex. Whether our two putative multimeric complexes are related to this ER complex or whether our complexes are characteristic of developing neural tissue in particular or of the wildtype ER is currently unknown.

Such an hypothesis is in no way meant to imply that estrogen activation of other substrates such as adenylate cyclase- (36,118) or Ca2+-dependent channels (119) or of other pathways (120), which also lead to phosphorylation of the ERKs via PKA/PKC (121) or via Ras (120), for example, may not also be occurring concurrently in the brain. It is also highly probable that there are influences from concurrent interactions of estrogen with other growth factors in the postnatal brain whose receptors, as tyrosine kinases, may also activate many of the same differentiative signaling cascades, utilized by the neurotrophins (e.g., the Ras/Raf-Map kinase cascade or SNT). On the other hand, other routes, involving the trk, PLC-y1/PKCa cascade, for example, which also phosphorylate and activate p21Ras (36,118,120,121) and thence the entire MAP kinase cascade may be important in recruiting and prolonging the initial rapid responses through the variety of pathways with which PKC interacts. And, because ER-containing cortical neurons also co-express the neurotrophin ligand mRNAs (13), it is entirely possible that the patterns of signaling we have observed may also reflect estrogen regulation of neurotrophin synthesis and release rather than direct activation of signaling intermediates. Such a mechanism may well explain estrogen regulation of genes such as NGF (122), BDNF (123) and the cholinergic enzymes, for example (124,125). However, our preliminary results document that, among its postulated CNS actions, estrogen does activate signaling proteins rapidly and directly, supporting the hypothesis of cross-coupling of the estrogen and neurotrophin signaling systems.

Because it has been suggested that complete activation of the ER requires both the intracytoplasmic and intranuclear action of at least six protein kinases (38), kinases such as ERK and Rsk, which translocate to the nucleus, may also act as signaling intermediates to phosphorylate the ER. Through nuclear translocation of ERK and Rsk, convergence or cross-coupling of the estrogen and neurotrophin signaling pathways to the nucleus, via the putative multimeric complexes and/or via individual components of the MAP kinase cascade, would provide alternative, novel, and unconventional routes to the nucleus to mediate estrogen and neurotrophin actions in the developing brain. Although the ligand-activated ER undoubtedly acts as a transcription factor, cross-coupling of estrogen and neurotrophin signalling pathways could explain how estrogen and the neurotrophins could each regulate the same broad array of ERE- and non-ERE-containing genes, involved in neuronal differentiation and neurite growth. For example, the neurite-and growth-related genes, ^-tubulin MAP-2, tau microtubule-associated protein, and GAP-43 are regulated not only by estrogen (126-129) but by the neurotrophins as well (85,130-134).

Whether the actions of NGF and the other neurotrophins in neural tissues are mediated only by trk receptors or also involve the ER as well is currently unknown. Evidence supporting each possibility has been reported in non-neural tumor cell lines. Cross-coupling of the ER with growth factor signaling pathways appears to be a general property of the ER. Estrogen-dependent and estrogen-independent interactions of the ER with neurotransmitters (e.g., dopamine) (32,33,135) and growth factor signaling pathways (e.g., EGF, IGF-I, insulin) (31,46,47,70,136,137) have been shown to activate the ER via the cell surface and to be implicated in the mediation of an increasing number of estrogen-induced/estrogen-like differentiative processes. For example, EGF reportedly interacts in the adult mouse uterus with both estrogen and EGF receptors independently of estrogen to elicit responses attributed to estrogen actions (44,46,69,71,137,138). Interdepen-dency of estrogen and growth factor (EGF, IGF-1) signaling pathways through the intermediary of MAP kinase has recently been implicated in the mediation of cell proliferation in non-neural estrogen targets, such as the uterus and tumor cell lines (138). IGF-1 and EGF caused phosphorylation of the unliganded ER on serine118, an action attributed to direct activation by MAP kinase (ERK) (31,46,47). On the other hand, Migliaccio et al. (65) have suggested that estradiol-induced phosphorylation of EGF and IGF-1 signaling pathways in MCF-7 cells requires ligand occupation of the classical ER, because the pure anti-estrogen ICI 182 780 inhibited estrogen activation (phosphorylation) of each component of the MAP kinase cascade. Moreover, very preliminary findings suggest that in the absence of estrogen the anti-estrogen ICI 164 384 blocked NGF phosphorylation of the ERKs completely (M. Singh and D. Toran-Allerand, unpublished observations), suggesting that in the brain both estrogen and neurotrophin receptors may be required for estrogen and neurotrophin activation of the ERKs and perhaps the upstream signaling proteins as well.

That estrogen and the neurotrophins each cause tyrosine phosphorylation and activation of the ER and the ERKs suggests multiple potential routes by which different signal transduction pathways may converge and influence one another. Cross-coupling of the estrogen and neurotrophin receptor systems may have great importance for the regulatory mechanisms underlying autocrine and paracrine responses in both the developing and adult brain. Convergence of the estrogen and neurotrophin signaling pathways may have relevance not only for the ontogeny of both the estrogen and neurotrophin receptor systems, but for neurodegenerative disorders and brain injury and the potential for regeneration and repair as well. Regenerative responses, which follow injury, also represent stages when functional neuronal contacts, ensuring target-derived (trans-synaptic) neurotrophin availability, have yet to be made. Thus, the ER may use multiple, possibly redundant, signaling pathways in mediating estrogen and neurotrophin responses, as has been shown for NGF and trkA in PC12 cells (139,140).

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