The classical mitogen-activated protein kinase (MAPK; also known as extracellular-signal-regulated kinase), ERK cascade has been shown to have a crucial role in cell proliferation and differentiation. In PC12 cells, sustained activation of ERK induced by nerve-growth factor (NGF) is essential for neuronal differentiation. However, downstream targets of ERK that are essential for neuronal differentiation have not been defined. Here we show that NGF induces strong, sustained expression of p35, the neuron-specific activator of cyclin-dependent kinase 5 (Cdk5), through activation of the ERK pathway. The induced kinase activity of Cdk5 is required for NGF-induced neurite outgrowth. Our results indicate that sustained activation of ERK is necessary and sufficient for strong induction of p35. Furthermore, the transcription factor Egr1, is induced by NGF through the ERK pathway and mediates induction of p35 by ERK. Our results thus define an essential signalling pathway, downstream of ERK/MAPK, that leads to neuronal differentiation.

In the mammalian central nervous system glutamate is the major excitatory neurotransmitter and plays a crucial role in plasticity and toxicity of certain neural cells. We found that glutamate stimulated activation of p38 and stress-activated protein kinase (SAPK, also known as c-Jun N-terminal kinase (JNK)), two subgroup members of the mitogen-activated protein kinase superfamily in matured cerebellar granule cells. The p38 activation was largely mediated by N-methyl-D-aspartate receptors. Furthermore, we have revealed a novel signaling pathway, that is, Ca 2؉ -mediated activation of p38 in glutamate-treated granule cells. The glutamate concentration effective for inducing apoptosis correlated with that for inducing p38 activation. SB203580, a specific inhibitor for p38, inhibited glutamate-induced apoptosis. Thus p38 might be involved in glutamateinduced apoptosis in cerebellar granule cells.

Nerve growth factor (NGF) induces sustained activation of classical MAP kinase (MAPK, also known as ERK) and neuronal differentiation in PC12 cells, whereas epidermal growth factor (EGF) induces transient activation of ERK/MAPK and stimulates proliferation of the cells. Although previous studies showed that sustained activation of ERK/MAPK is important for neuronal differentiation of the cells, a recent report revealed that inhibition of the sustained phase of ERK/MAPK activation alone does not block neurite outgrowth caused by NGF. These results suggest requirement for an additional signaling pathway(s) triggered by NGF in neuronal differentiation. Here we show that NGF induces sustained activation of p38, a subfamily member of the MAPK superfamily, and that inhibition of the p38 pathway blocks neurite outgrowth in PC12 cells. Surprisingly, expression of constitutively active MAPK/ERK kinase (MAPKK, also known as MEK) results in p38 activation as well as ERK/MAPK activation, and a p38 inhibitor blocks neurite outgrowth caused by the constitutively active MAPKK/MEK. Moreover, constitutive activation of p38 is able to induce neurite outgrowth when combined with EGF treatment. These results reveal an essential role of p38 in neuronal differentiation in PC12 cells.The phaeochromocytoma cell line PC12 is a well studied model of actions of neurotrophic factors such as NGF 1 (1, 2). NGF treatment of PC12 cells leads to their differentiation into sympathetic-like neurons characterized by neurite outgrowth. Previous studies have suggested that NGF-induced, sustained activation of the ERK/MAPK pathway (3-6) is crucial for neuronal differentiation of the cells, since blockade of the ERK/ MAPK activation inhibits neurite induction, and constitutive activation of the ERK/MAPK pathway results in neurite outgrowth (7,8). However, a recent finding that although the sustained activation of ERK/MAPK results from activation of Rap1, expression of a mutant Rap1 that blocks the sustained phase of ERK/MAPK activation does not inhibit neurite outgrowth triggered by NGF demonstrates that neurite outgrowth by NGF does not require sustained activation of ERK/MAPK (9). Moreover, there was another finding that bone morphogenetic protein-2 is able to induce neuronal differentiation of PC12 cells without marked activation of ERK/MAPK (10). Bone morphogenetic protein-2 was also reported to induce neuronal differentiation in neural crest stem cells (11). These observations suggest the existence of an additional signaling pathway(s) important for neuronal differentiation of PC12 cells. On one hand, we observed activation of p38 as well as ERK/MAPK in response to brain-derived neurotrophic factor, a member of an NGF family of neurotrophic factors, in cultured cerebellar granule cells. 2 We therefore examined a possible involvement of p38 in NGF actions. We have shown that NGF treatment induces rapid and relatively long activation of p38 and that inhibition of p38 by a specific inhibitor SB203580 or by expression of dominant-negative constructs ...

Purpose: Akt is a serine/threonine kinase that plays a central role in tumorigenesis. Among the members of Akt family, Akt2 is associated with the development of human cancers. The present study was designed to clarify the prognostic significance of Akt2 and activated Akt expression in pancreatic ductal adenocarcinoma (PDAC). In addition, activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) and the proliferation activity of tumor cells detected by Ki-67 immunohistochemistry were examined.Experimental Design: Immunohistochemical analysis was performed on paraffin-embedded specimens from 65 patients with PDAC; 36 males and 29 females with ages ranging from 48 to 79 years (median, 66 years) of age. Expression levels of Akt2, phosphorylated Akt (p-Akt), and phosphorylated ERK 1/2 (p-ERK 1/2) were categorized as either weaker (low intensity) or equal to stronger (high intensity) compared with those in the endothelial cells of the same specimens. For Ki-67 immunohistochemistry, cases were divided into two groups: level 1, Ki-67 labeling index (LI), <20%; level 2, Ki-67 LI, >20%.Results: Twenty-six (42.6%), 28 (45.9%), 39 (63.9%), and 46 (75.4%) of the tumors showed high intensity of Akt2, p-Akt, and p-ERK 1/2 expression, and Ki-67 LI level 2, respectively. A significant positive correlation was observed between Akt2 and p-Akt expression (P < 0.01). Multivariate analysis revealed that p-Akt expression, Ki-67 LI, and histological differentiation are independent prognosticators for PDAC.Conclusions: p-Akt expression is a significant prognostic indicator for PDAC. Inhibition of Akt is a possible molecular approach for treatment of PDAC.

Cell gene expression is affected by both the kind and mode of growth factor stimulation (diffusive vs. non-diffusive). Epidermal growth factor (EGF) was pattern-immobilized on a polystyrene plate. Although the growth of the rat phaeochromocytoma cell line PC12 is stimulated by diffusible EGF, and differentiation is stimulated by diffusible nerve growth factor (NGF), immobilized (non-diffusible) EGF stimulated PC12 differentiation. The immobilized EGF caused a long-lasting stimulation of the intracellular signal protein mitogen-associated protein MAP kinase (MAPK, also known as ERK) and p38 (a subfamily of the MAPK superfamily) in cells, as did diffusible NGF. The switching between growth stimulation and differentiation is considered to be due to the duration of the stimulus. The function of the biosignal conjugate was regulated using conjugation methodology.

Purpose: AKT is a serine/threonine kinase which is important in tumorigenesis. Several molecules involved in AKT pathway are dysregulated in various kinds of human cancers. Patients and Methods: Ninety-three patients (53 males and 40 females), ages ranging from 19 to 77 years (median, 57 years), with localized soft-tissue sarcomas arising in the trunk and extremities, were analyzed. Immunoperoxidase procedure (avidin-biotin complex method) was done on paraffin-embedded sections with anti^phosphorylated AKT (Thr 308

The mitogen-activated protein kinase (MAPK) cascade has been shown to play an essential role in regulation of cell proliferation and cell differentiation. Although mammalian MAPKs are most abundantly expressed in postmitotic and terminally differentiated neuronal cells, their function in the central nervous system is still largely undefined. We present evidence here for a role of the MAPK cascade in cerebellar long term depression (LTD), which is a widely studied form of synaptic plasticity in mammalian brain. In cultured Purkinje cells, LTD is known to be induced by iontophoretic application of glutamate and depolarization of Purkinje cells. We found that MAPK was activated in Purkinje cells by treatment of primary cultures of rat embryonic cerebella with glutamate and a depolarization-inducing agent, KCl. Application of PD98059, a specific inhibitor of MAPK kinase (MAPKK/MEK), inhibited both the activation of MAPK and the induction of LTD in Purkinje cells. Furthermore, the induction of LTD was completely blocked by introduction into Purkinje cells of anti-active MAPK antibody, which was found to specifically and potently inhibit the activity of MAPK. These results suggest that postsynaptic activation of the MAPK cascade is essential for the induction of cerebellar LTD.Mitogen-activated protein kinase (MAPK, 1 also known as ERK) is a serine/threonine protein kinase that is commonly activated by various growth factors and differentiation factors (1-3). Activation of MAPK requires phosphorylation of both threonine and tyrosine residues in its TEY sequence, which is catalyzed by an upstream activator MAPK kinase (MAPKK, also known as MEK). MAPK and MAPKK constitute a functional unit called the MAPK cascade, which has been shown to play a crucial role in regulation of cell proliferation, cell differentiation, and early embryonic development. The abundant expression of both MAPK and MAPKK in postmitotic and differentiated neurons (1-5), however, suggests a possible function of the MAPK cascade in the mammalian central nervous system.Persistent changes in synaptic strength such as hippocampal long term potentiation and cerebellar long term depression (LTD) are thought to be cellular mechanisms for learning and memory (6, 7). Cerebellar LTD is a persistent reduction of synaptic transmission between parallel fibers and Purkinje cells (7,8). Cerebellar LTD is elicited by the simultaneous activation of parallel fibers and climbing fibers, which can be replaced by iontophoretic application of glutamate and depolarization of Purkinje cells, respectively. Although the induction of cerebellar LTD has been reported to require an increase in intracellular Ca 2ϩ concentration and protein kinase C in Purkinje cells (9 -12), molecular mechanisms of the induction of cerebellar LTD are not well defined. It has previously been reported that treatment of cultured cortical neurons with glutamate, which triggers Ca 2ϩ influx through the N-methyl-Daspartate type of glutamate receptors, results in activation of MAPK (13). In marine snai...

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