Regulation and role of cyclin-dependent kinase activity in neuronal survival and death

Cyclin-dependent kinase (Cdk)5 is a proline-directed Ser/Thr protein kinase that functions mainly in neurons and is activated by binding to a regulatory subunit, p35 or p39. Kinase activity is mainly determined by the amount of p35 available, which is controlled by a balance between synthesis and degradation. Kinase activity is also regulated by Cdk5 phosphorylation, but the activity of phosphorylated Cdk5 is in contrast to that of cycling Cdks. Cdk5 is a versatile protein kinase that regulates multiple neuronal activities including neuronal migration and synaptic signaling. Further, Cdk5 plays a role in both survival and death of neurons. Long-term inactivation of Cdk5 triggers cell death, and the survival activity of Cdk5 is apparent when neurons suffer from stress. In contrast, hyper-activation of Cdk5 by p25 promotes cell death, probably by reactivating cell-cycle machinery in the nucleus. The pro-death activity is suppressed by membrane association of Cdk5 via myristoylation of p35. Appropriate activity, localization, and regulation of Cdk5 may be critical for long-term survival of neurons, which is more than 80 years in the case of humans.     

Cytoskeletal reorganization mediates fluid shear stress-induced ERK5 activation in osteoblastic cells

Mechanotransduction is a complicated process, of which mechanosensation is the first step. Previous studies have shown that the cytoskeleton plays a crucial role in mechanosensation and the mediation of intracellular signal transduction. However, the mechanism of mechanotransduction in the bone remains elusive. Here, we investigated the potential involvement of a novel MAPK (mitogen-activated protein kinase) member, ERK5 (extracellular-signal-regulated kinase 5), in the response of osteoblastic cells to FSS (fluid shear stress). Our results demonstrated that ERK5 was rapidly phosphorylated in pre-osteoblastic MC3T3-E1 cells upon FSS, and the integrity and reorganization of the cytoskeleton were critical in this process, in which the cytoskeleton-dependent activation of FAK (focal adhesion kinase) may be involved in the activation of ERK5 induced by FSS. Moreover, we found that cytoskeletal disruption led to significant down-regulation of ERK5 phosphorylation, but had no effect on ERK5 nuclear localization. Furthermore, the cytoskeleton rapidly reorganized in response to FSS, but long-time fluid load, even at a physiological level, led to cytoskeletal disruption, suggesting that other pathways may be involved in long-term mechanotransduction. Taken together, our data provide new insight into the mechanisms of mechanosensation by highlighting the link between ERK5 activation and cytoskeletal reorganization in osteoblasts undergoing FSS.     

Cdk5 regulates multiple cellular events in neural development,function and disease

Cyclin‐dependent kinases (CDKs) generally regulate cell proliferation in dividing cells, including neural progenitors. In contrast, an unconventional CDK, Cdk5, is predominantly activated in post‐mitotic cells, and involved in various cellular events, such as microtubule and actin cytoskeletal organization, cell–cell and cell–extracellular matrix adhesions, and membrane trafficking. Interestingly, recent studies have indicated that Cdk5 is associated with several cell cycle‐related proteins, Cyclin‐E and p27^kip1. Taking advantage of multiple functionality, Cdk5 plays important roles in neuronal migration, layer formation, axon elongation and dendrite arborization in many regions of the developing brain, including cerebral cortex and cerebellum. Cdk5 is also required for neurogenesis at least in the cerebral cortex. Furthermore, Cdk5 is reported to control neurotransmitter release at presynaptic sites, endocytosis of the NMDA receptor at postsynaptic sites and dendritic spine remodeling, and thereby regulate synaptic plasticity and memory formation and extinction. In addition to these physiological roles in brain development and function, Cdk5 is associated with many neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. In this review, I will introduce the physiological and pathological roles of Cdk5 in mammalian brains from the viewpoint of not only in vivo phenotypes but also its molecular and cellular functions.     

Ginsenosides promote proliferation of granulosa cells from chicken prehierarchical follicles through PKC activation and up‐regulated cyclin gene expression

The effect of GS (ginsenosides) on proliferation of chicken GCs (granulosa cells) from prehierarchical SYF (small yellow follicles) was evaluated, and involvement of the PKC (protein kinase C) signalling pathway as well as mRNA expression of cyclins and CDK (cyclin‐dependent kinase) were investigated. Whole SYF or GCs isolated from SYF were cultured in Medium 199 supplemented with 0.5% FCS (fetal calf serum). After 16 h, the cells were challenged with GS alone or in combination with PKC inhibitor H₇ or activator PMA (phorbol 12‐myristate 13‐acetate) for 24 h in serum‐free medium. Results showed that in both whole follicles and pure GCs monolayer culture system, GS (0.1–10 μg/ml) significantly increased the number of GCs in SYF in a dose‐dependent manner, and this stimulatory effect was inhibited by H₇, but enhanced by PMA. Meanwhile, the PCNA‐LI (proliferating cell nuclear antigen labelling index) of GCs displayed similar changes with the cell number. Mechanism of GS action was further evaluated in cultured GCs separated from SYF. Western blot analysis showed that 10 μg/ml GS increased PKC translocation from cytoplasm to the plasma membrane of the GCs to become the active state. This effect was blocked by H₇. Furthermore, GS up‐regulated the expression of cyclin D1/CDK6 and cyclin E/CDK2 mRNAs in GCs; however, inhibition of PKC with H₇ attenuated this stimulatory effect. These results indicated that GS could stimulate proliferation of chicken GCs through activated PKC‐involved up‐regulation of cyclin D1/CDK6 and cyclin E/CDK2 genes, subsequently promoting development of the chicken prehierarchical follicles.     

Depolarization-induced ERK phosphorylation depends on the cytosolic Ca2+ level rather than on the Ca2+ channel subtype of chromaffin cells

The contribution of Ca2+ entry through different voltage-activated Ca2+ channel (VACC) subtypes to the phosphorylation of extracellular signal regulated kinase (ERK) was examined in bovine adrenal-medullary chromaffin cells. High K+ depolarization (40 mM, 3 min) induced ERK phosphorylation, an effect that was inhibited by specific mitogen-activated protein kinase kinase inhibitors. By using selective inhibitors, we observed that depolarization-induced ERK phosphorylation completely depended on protein kinase C-alpha (PKC-alpha), but not on Ca2+/calmodulin-dependent protein kinase nor cyclic AMP-dependent protein kinase. Blockade of L-type Ca2+ channels by 3 microm furnidipine, or blockade of N channels by 1 micromomega-conotoxin GVIA reduced ERK phosphorylation by 70%, while the inhibition of P/Q channels by 1 micromomega-agatoxin IVA only caused a 40% reduction. The simultaneous blockade of L and N, or P/Q and N channels completely abolished this response, yet 23% ERK phosphorylation remained when L and P/Q channels were simultaneously blocked. Confocal imaging of cytosolic Ca2+ elevations elicited by 40 mm K+, showed that Ca2+ levels increased throughout the entire cytosol, both in the presence and the absence of Ca2+ channel blockers. Fifty-eight percent of the fluorescence rise depended on Ca2+ entering through N channels. Thus, ERK phosphorylation seems to depend on a critical level of Ca2+ in the cytosol rather than on activation of a given Ca2+ channel subtype.     

Integrin-mediated adhesion regulates ERK nuclear translocation and phosphorylation of Elk-1

Integrin-mediated adhesion to the extracellular matrix permits efficient growth factor-mediated activation of extracellular signal-regulated kinases (ERKs). Points of regulation have been localized to the level of receptor phosphorylation or to activation of the downstream components, Raf and MEK (mitogen-activated protein kinase/ERK kinase). However, it is also well established that ERK translocation from the cytoplasm to the nucleus is required for G1 phase cell cycle progression. Here we show that phosphorylation of the nuclear ERK substrate, Elk-1 at serine 383, is anchorage dependent in response to growth factor treatment of NIH 3T3 fibroblasts. Furthermore, when we activated ERK in nonadherent cells by expression of active components of the ERK cascade, subsequent phosphorylation of Elk-1 at serine 383 and Elk-1-mediated transactivation were still impaired compared with adherent cells. Elk-1 phosphorylation was dependent on an intact actin cytoskeleton, as discerned by treatment with cytochalasin D (CCD). Finally, expression of active MEK failed to predominantly localize ERK to the nucleus in suspended cells or adherent cells treated with CCD. These data show that integrin-mediated organization of the actin cytoskeleton regulates localization of activated ERK, and in turn the ability of ERK to efficiently phosphorylate nuclear substrates.     

Differential regulation of basic protein phosphorylation by calcium phospholipid and cyclic-AMP-dependent protein kinases

Myelin basic protein, an 80-kilodalton (kDa) protein in rat oligodendrocytes, and an 80-kDa basic protein in neuroblastoma x neonatal Chinese hamster brain explant hybrids were phosphorylated extensively when the cells were treated with either phorbol esters (TPA) or diacylglycerols (e.g., oleyoyl-acetylglycerol). TPA-stimulated phosphorylation was inhibited by pre-incubation with 50 microM psychosine (galactosyl-sphingosine), confirming that it is mediated through the phospholipid-dependent protein kinase C (PK-C). Surprisingly, phosphorylation of these proteins was inhibited by incubation of cells with agents which result in activation of cyclic-AMP-dependent protein kinase (dibutyryl cyclic AMP or forskolin). In contrast, phosphorylation of other nonbasic proteins, for example, the oligodendrocyte-specific 2',3'-cyclic nucleotide phosphohydrolase, was stimulated under these conditions (Vartanian et al.: Proceedings of the National Academy of Sciences of the United States of America 85:939, 1988). The possible role of cyclic AMP in activating specific phosphatases or restricting the availability of diacylglycerol for PK-C activation is discussed.     

Agonist-induced internalization of histamine H2 receptor and activation of extracellular signal-regulated kinases are dynamin-dependent

Histamine H2 receptor (H2R) is a member of G protein-coupled receptor family. Agonist stimulation of H2R results in several cellular events including activation of adenylate cyclase and phospholipase C, desensitization of the receptor, activation of extracellular signal-regulated kinases ERK1/2, and receptor endocytosis. In this study, we identified a GTPase dynamin as a binding partner of H2R. Dynamin could associate with H2R both in vitro and in vivo. Functional analyses using dominant-negative form of dynamin (K44E-dynamin) revealed that cAMP production and the following H2R desensitization are independent of dynamin. However, the agonist-induced H2R internalization was inhibited by co-expression of K44E-dynamin. Furthermore, activation of extracellular-signal regulated kinases ERK1/2 in response to dimaprit, an H2R agonist, was attenuated by K44E-dynamin. Although H2R with truncation of 51 amino acids at its carboxy-terminus did not internalize after agonist stimulation, it still activated ERK1/2, but the degree of this activation was less than that of the wild-type receptor. Finally, K44E dynamin did not affect ERK1/2 activation induced by internalization-deficient H2R. These results suggest that the agonist-induced H2R internalization and ERK1/2 activation are partially dynamin-dependent. Furthermore, ERK1/2 activation via H2R is likely dependent of the endocytotic process rather than dynamin itself.     

The regulation of ERK and p-ERK expression by cisplatin and sorafenib in gastric cancer cells

Previous studies have reported strong antitumor effects of cisplatin and sorafenib. Our results indicated that cisplatin and sorafenib exhibited anti-tumor effects on gastric cancer cells. They significantly inhibited gastric cell growth and induced apoptosis. They effectively inhibited gastric cancer cell proliferation and induced G0/G1 phase arrest. Western blotting analysis indicated that it also promoted the phosphorylation extracellular signal regulated kinase (p-ERK). Moreover, cisplatin and sorafenib played a synergistic antitumor effect. These results suggested that the antitumor mechanism of cisplatin and sorafenib involved altering the cell cycle and stimulating ERK phosphorylation in the ERK signaling pathway.     

Serum regulates adipogenesis of mesenchymal stem cells via MEK/ERK‐dependent PPARγ expression and phosphorylation

Mesenchymal stem cells (MSCs) provide us an excellent cellular model to uncover the molecular mechanisms underlying adipogenic differentiation of adult stem cells. PPARγ had been considered as an important molecular marker of cells undergoing adipogenic differentiation. Here, we demonstrated that expression and phosphorylation of PPARγ could be found in bone marrow–derived MSCs cultured in expansion medium without any adipogenic additives (dexamethasone, IBMX, insulin or indomethacin). Then, PPARγ was dephosphorylated in MSCs during the process of adipogenic differentiation. We then found that inhibition of MEK activation by specific inhibitor (PD98059) counteracted the PPARγ expression and phosphorylation. However, expression and phosphorylation of PPARγ did not present in MSCs cultured in medium with lower serum concentration. When these MSCs differentiated into adipocytes, no phosphorylation could be detected to accompany the expression of PPARγ. Moreover, exposure of MSCs to higher concentration of serum induced stronger PPARγ expression, and subsequently enhanced their adipogenesis. These data suggested that activation of the MEK/ERK signalling pathway by high serum concentration promoted PPARγ expression and phosphorylation, and subsequently enhanced adipogenic differentiation of MSCs.     

Identification of p10 as a neurotoxic product generated from the proteolytic cleavage of the neuronal Cdk5 activator

The involvement of cyclin‐dependent kinase‐5 (Cdk5) and p25, the proteolytic fragment of activator p35, has long been implicated in the development of neuron‐fibrillary tangles (NFTs), a hallmark of Alzheimer's disease (AD). Findings in this area over the past decade have been highly controversial and inconclusive. Here we report unprecedented detection of endogenous p10, the smaller proteolytic fragment of the Cdk5 activator p35 in treated primary cortical neurons that underwent significant apoptosis, triggered by proteasome inhibitors MG132 and lactacystin, and protein kinase inhibitor staurosporine (STS). p10 appeared exclusively in the detergent‐resistant fraction made up of nuclear matrix, membrane‐bound organelles, insoluble membrane proteins, and cytoskeletal components. Intriguingly, transient overexpression of p10 in neural cells induced apoptotic morphologies, suggesting that p10 may play an important role in mediating neuronal cell death in neurodegenerative diseases. We demonstrated for the first time that p10‐mediated apoptosis occurred via a caspases‐independent pathway. Furthermore, as p10 may contain the myristoylation signal for p35 which is responsible for binding p35 to several intracellular components and the membrane, all in all these novel results present that the accumulation of p10 to the detergent‐insoluble fraction may be a crucial pathological event to triggering neuronal cell death. J. Cell. Biochem. 111: 1359–1366, 2010. © 2010 Wiley‐Liss, Inc.