Cyclin-dependent kinase 5 (Cdk5) in Physiology and Pathology

细胞周期蛋白依赖性激酶5 （Cdk5）主要在神经系统中表达。虽然它在非神经元细胞中表达，并与细胞周期蛋白结合，但由于其与神经元特异性分子P35和P39结合，其活性主要存在于有丝分裂后的神经元中。它参与神经元迁移、突触传递和存活。Cdk5是一种多功能神经元激酶（靶向从神经元分化到突触功能的蛋白质），当与其共激活因子p35络合时受到严格调控。它是磷酸化神经丝和tau蛋白的几种激酶之一。它的多种作用部分源于其在神经细胞存活、生长和分化的信号转导网络中与其他激酶的串扰相互作用。例如，我们已经证明，Cdk5下调mapk和jnk，上调pi3k。这些结果表明，Cdk5通常调节其他激酶对神经元存活的特定信号的反应强度。Cdk5在突触传递中的作用是通过调节突触前蛋白（如synapsin、Munc18和dynamin 1）的细胞功能来介导的。它在突触中的多功能作用是复杂的，可能涉及其他新的底物。为了探索这种可能性，我们使用酵母双杂交筛选人类cDNA文库，以p35为诱饵，分离出人类SEPT5 (SEPT5)，也称为hCDCrel-1，作为相互作用克隆。Cdk5及其激活剂p35在GST（谷胱甘肽s -转移酶）下拉和共免疫沉淀试验中都与SEPT5相关。我们证实了Cdk5/p35在体外和体内磷酸化SEPT5，并确定了SEPT5的S327位点是一个主要的磷酸化位点。一个丝氨酸(S)-丙氨酸(A) 327突变体比SEPT5野生型更有效地结合syntaxin。此外，来自突触囊泡组分和Cdk5野生型和敲除裂解物的共免疫沉淀表明，Cdk5/p35磷酸化septin 5降低了其与syntaxin-1的结合。此外，突变型非磷酸化SEPT5在PC12细胞中表达时，比野生型更能增强受调节的胞吐作用。这些数据表明Cdk5在S327位点磷酸化人SEPT5在调节胞外分泌中起作用。

Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in the nervous system. Though it is expressed in non- neuronal cell and binds with cyclins but its activity is predominantly found in post mitotic neurons due to its binding to neuron specific molecules P35 and P39. It is involved in neuronal migration, synaptic transmission, and survival.Cdk5,a multifunctional neuronal kinase (targeting proteins from neuronal differentiation to synaptic function), is tightly regulated when complexed with p35, its co-activator. It is one of several kinases that phosphorylate neurofilaments and tau. Its diverse roles stem, in part, from its cross-talk interactions with other kinases in signal transduction networks underlying neuronal cell survival, growth and differentiation. We have shown, for example, that Cdk5 down regulates MAPKs and JNKs and up regulates PI3Ks. These results suggest that Cdk5 normally modulates the intensity of response of other kinases to specific signals underlying neuronal survival. The role of Cdk5 in synaptic transmission is mediated by regulating the cellular functions of presynaptic proteins such as synapsin, Munc18, and dynamin 1. Its multifunctional role at the synapse is complex and probably involves other novel substrates. To explore this possibility, we used a yeast two-hybrid screen of a human cDNA library with p35 as bait and isolated human septin 5 (SEPT5), known also as hCDCrel-1, as an interacting clone. Both,Cdk5 and its activator, p35 associates with SEPT5 in GST (glutathione S-transferase)-pull-down and coimmunoprecipitation assays. We confirmed that Cdk5/p35 phosphorylates SEPT5 in vitro and in vivo and identified S327 of SEPT5 as a major phosphorylation site. A serine (S)-to-alanine (A) 327 mutant of SEPT5 bound syntaxin more efficiently than SEPT5 wild type. Additionally, coimmunoprecipitation from synaptic vesicle fractions and Cdk5 wild-type and knock-out lysates showed that phosphorylation of septin 5 by Cdk5/p35 decreases its binding to syntaxin-1. Moreover, mutant nonphosphorylated SEPT5 potentiated regulated exocytosis more than the wild type when each was expressed in PC12 cells. These data suggest that Cdk5 phosphorylation of human septin SEPT5 at S327 plays a role in modulating exocytotic secretion. When deregulated, however, in stressed neurons, the p35 activator is cleaved by proteases to a truncated fragment, p25, that binds and hyperactivates Cdk5; this abnormal Cdk5/p25 complex contributes to the pathology seen in several neurodegenerative diseases. Most therapeutic approaches targeting the deregulated hyperactive Cdk5/p25 complex, as well as other kinases implicated in neurodegenerative pathology, inhibit activity by interfering with the kinase ATP binding domain. Many drugs like roscovitine, which binds to the Cdk5 ATP site, have been evaluated but lack the specificity required since most cdk kinases (and most kinases) are vulnerable at the ATP binding site. Our study of site specific interactions between Cdk5 and truncated forms of its p35 regulator have revealed a central fragment, 125 amino acids residues (CIP) that has high affinity for and inhibits the in vitro and in situ activity of the Cdk5/p25 complex. We have shown that CIP specifically inhibits Cdk5/p25 (deregulated) activity in transfected cells and also reduces the aberrant and hyperphosphorylation of tau and neurofilament proteins in stressed neurons. It is important to note that CIP does not affect the activity of regulated Cdk5 ( Cdk5/P35). Recently, we have found a much smaller truncated peptide of 24 amino acids derived from p35 that also specifically inhibits Cdk5-deregulated activity. This may provide a possible new and novel therapeutic route for intervention to prevent or reduce the neurodegenerative pathology induced by Cdk5 deregulation. Recently p35 expression and cdk5 activity have been reported in pancreatic beta cells. Chronic exposure to gllucose, as in hyperglycemia, results in hyperactivity of Cdk5 and reduced insulin secretion. Inhibition of Cdk5 activity increases insulin secretion under conditions of high but not low glucose. These studies suggest that inhibition of cdk5/p35 activity may overcome the risks of hypoglycemic episodes induced by the therapeutic use of sulfonylurea for type 2 diabetes. In amyloid stressed neurons we found that p25, a truncated fragment of p35, accumulates and hyperactivates cdk5 and induces apoptosis. Similarly, amyloid deposition within the insulin producing islets of Langerhans in the pancreas, is also pathological. Accordingly, in a recent study we demonstrated that abnormal upregulation of p25 and cdk5/p25 activity induced in a pancreatic beta cell line, resulted in 70% inhibition of insulin secretion in low and high glucose. We also showed that CIP, which inhibits Cdk5/ p25 activity without affecting Cdk5/p35 activity in neurons also inhibited Cdk5/p25 activity in MIN 6 pancreatic cells and in turn, increased insulin secretion. Significantly, as in neurons, CIP had no effect on endogenous Cdk5/p35 activity and insulin secretion. Co-infection of dominant negative Cdk5 (DNCdk5) with p35 or p25 inhibited aberrant activation of Cdk5 activity and insulin secretion levels were increased. These studies indicate that over-activation of Cdk5 deregulates insulin secretion and induces cell death in pancreatic beta cells and suggests that CIP may serve as a therapeutic agent for type 2 diabetes. Our results are consistent with the view that AD and type 2 diabetes mellitus (T2DM) are linked metabolically and pathologically in a number of ways. They share such abnormalities as impaired glucose metabolism, increased oxidative stress, amyloid deposition and insulin resistance. The deposition of amyloid within the insulin-producing islets of Langerhans in the pancreas is a common pathological finding in patients with T2DM. Moreover, it has been demonstrated that T2DM is associated with an increased risk of cognitive dysfunction and dementia. Our results, however. go much further in establishing the similarity between neuron and insulin secreting beta cells; they show that deregulation of cdk5 in pancreatic cells follows the pattern of cdk5 deregulation in neurodegenerative disorders and in both cases CIP effectively prevents the pathology and promotes cell survival. These dramatic results are consistent with the suggestion that cdk5 may be a therapeutic target for the treatment of diabetes mellitus and certain neurodegenerative disorders in which cdk5 is deregulated.