Phosphorylation of spinophilin by ERK and cyclin-dependent PK 5 (Cdk5)

Spinophilin is a protein that binds to protein phosphatase-1 and actin and modulates excitatory synaptic transmission and dendritic spine morphology. We have identified three sites phosphorylated by ERK2 (Ser-15 and Ser-205) and cyclin-dependent PK 5 (Cdk5) (Ser-17), within the actin-binding domain of spinophilin. Cdk5 and ERK2 both phosphorylated spinophilin in intact cells. However, in vitro, phosphorylation by ERK2, but not by Cdk5, was able to modulate the ability of spinophilin to bind to and bundle actin filaments. In neurons and HEK293 cells expressing GFP-tagged variants of spinophilin, imaging studies demonstrated that introduction of a phospho-site mimic (Ser-15 to glutamate) was associated with increased filopodial density. These results support a role for spinophilin phosphorylation by ERK2 in the regulation of spine morphogenesis.     

Regulation of cyclin-dependent kinase 5 catalytic activity by phosphorylation

Cyclin-dependent kinase 5 (cdk5) is found in an active form only in neuronal cells. Activation by virtue of association with the cyclin-like neuronal proteins p35 (or its truncated form p25) and p39 is the only mechanism currently shown to regulate cdk5 catalytic activity. In addition to cyclin binding, other members of the cdk family require for maximal activation phosphorylation of a Ser/Thr residue (Thr(160) in the case of cdk-2) that is conserved in all cdks except cdk8. This site is phosphorylated by cdk-activating kinases, which, however, do not phosphorylate cdk5. To examine the possible existence of a phosphorylation-dependent regulatory mechanism in the case of cdk5, we have metabolically labeled PC12 cells with (32)P(i) and shown that the endogenous cdk5 is phosphorylated. Bacterially expressed cdk5 also can be phosphorylated by PC12 cell lysates. Phosphorylation of cdk5 by a PC12 cell lysate results in a significant increase in cdk5/p25 catalytic activity. Ser(159) in cdk5 is homologous to the regulatory Thr(160) in cdk2. A Ser(159)-to-Ala (S159A) cdk5 mutant did not show similar activation, which suggests that cdk5 is also regulated by phosphorylation at this site. Like other members of the cdk family, cdk5 catalytic activity is influenced by both p25 binding and phosphorylation. We show that the cdk5-activating kinase (cdk5AK) is distinct from the cdk-activating kinase (cyclin H/cdk7) that was reported previously to neither phosphorylate cdk5 nor affect its activity. We also show that casein kinase I, but not casein kinase II, can phosphorylate and activate cdk5 in vitro.     

Regulation of synaptojanin 1 by cyclin-dependent kinase 5 at synapses

Synaptojanin 1 is a polyphosphoinositide phosphatase concentrated in presynaptic nerve terminals, where it dephosphorylates a pool of phosphatidylinositol 4,5-bisphosphate implicated in synaptic vesicle recycling. Like other proteins with a role in endocytosis, synaptojanin 1 undergoes constitutive phosphorylation in resting synapses and stimulation-dependent dephosphorylation by calcineurin. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates synaptojanin 1 and regulates its function both in vitro and in intact synaptosomes. Cdk5 phosphorylation inhibited the inositol 5-phosphatase activity of synaptojanin 1, whereas dephosphorylation by calcineurin stimulated such activity. The activity of synaptojanin 1 was also stimulated by its interaction with endophilin 1, its major binding partner at the synapse. Notably, Cdk5 phosphorylated serine 1144, which is adjacent to the endophilin binding site. Mutation of serine 1144 to aspartic acid to mimic phosphorylation by Cdk5 inhibited the interaction of synaptojanin 1 with endophilin 1. These results suggest that Cdk5 and calcineurin may have an antagonistic role in the regulation of synaptojanin 1 recruitment and activity, and therefore in the regulation of phosphatidylinositol 4,5-bisphosphate turnover at synapses.     

Regulation of cyclin-dependent kinase 5 and casein kinase 1 by metabotropic glutamate receptors

Cyclin-dependent kinase 5 (Cdk5) is a multifunctional neuronal protein kinase that is required for neurite outgrowth and cortical lamination and that plays an important role in dopaminergic signaling in the neostriatum through phosphorylation of Thr-75 of DARPP-32 (dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa). Casein kinase 1 (CK1) has been implicated in a variety of cellular functions such as DNA repair, circadian rhythm, and intracellular trafficking. In the neostriatum, CK1 has been found to phosphorylate Ser-137 of DARPP-32. However, first messengers for the regulation of Cdk5 or CK1 have remained unknown. Here we report that both Cdk5 and CK1 are regulated by metabotropic glutamate receptors (mGluRs) in neostriatal neurons. (S)-3,5-dihydroxyphenylglycine (DHPG), an agonist for group I mGluRs, increased Cdk5 and CK1 activities in neostriatal slices, leading to the enhanced phosphorylation of Thr-75 and Ser-137 of DARPP-32, respectively. The effect of DHPG on Thr-75, but not on Ser-137, was blocked by a Cdk5-specific inhibitor, butyrolactone. In contrast, the effects of DHPG on both Thr-75 and Ser-137 were blocked by CK1-7 and IC261, specific inhibitors of CK1, suggesting that activation of Cdk5 by mGluRs requires CK1 activity. In support of this possibility, the DHPG-induced increase in Cdk5 activity, measured in extracts of neostriatal slices, was abolished by CK1-7 and IC261. Treatment of acutely dissociated neurons with DHPG enhanced voltage-dependent Ca(2+) currents. This enhancement was eliminated by either butyrolactone or CK1-7 and was absent in DARPP-32 knockout mice. Together these results indicate that a CK1-Cdk5-DARPP-32 cascade may be involved in the regulation by mGluR agonists of Ca(2+) channels.     

CDK5在肝细胞癌中的异常表达

目的:研究周期素依赖性激酶5(cyclin-dependent kinase5.CDK5)在肝细胞癌(hepatocellular carcinoma,HCC)发生发展中的异常表达.方法:应用反转录聚合酶链式反应(RT-PCR)检测CDK5 mRNA的表达水平;免疫组织化学技术检测CDK5蛋白在正常肝组织、肝硬化和HCC组织中的表达:分析CDK5与临床病理学特征的关系.结果:CDK5 mRNA在HCC中呈上调表达,阳性表达率在正常肝组织、肝硬化和HCC组织中分别为35.3%、64.3%、89.7%;正常肝组织与HCC组织有显著差异(P<0.05),肝硬化组织与HCC组织也有显著差异(P<0.01).CDK5蛋白在HCC中高表达,阳性表达率在人正常肝组织、肝硬化和HCC组织之间分别为29.4%.64.3%,89.7%,三组之间差异有统计学意义(x2=58.095,P<0.01).HCC组织中CDK5蛋白表达强度与肿瘤的病理学分级显著相关(x2=19.330,P<0.01).结论:CDK5在HCC中上调表达,他的异常表达在HCC的发生发展过程中发挥重要的作用,且与肿瘤的分化程度相关.     

Loss-of-SIRT1 function during vascular ageing: Hyperphosphorylation mediated by cyclin-dependent kinase 5

The longevity regulator SIRT1 is an enzyme catalyzing the deacetylation of protein substrates, in turn modulating their biological functions. In endothelial cells, downregulation of SIRT1 evokes cellular senescence. In aged arteries, SIRT1 expression and activity is blunted, which contributes to the development of atherosclerosis and abnormal vascular responses. A recent study suggests that cyclin-dependent kinase 5 (CDK5) is responsible for the phosphorylation of SIRT1 at the serine 47 residue. This modification blocks the anti-senescence activity of SIRT1 and plays a critical role in the loss-of-SIRT1 function during vascular ageing. Thus, by inhibiting CDK5, SIRT1 function can be improved, in turn preventing the development of atherosclerosis and slowing down the process of vascular ageing.     

Phosphorylation and inactivation of protein phosphatase 1 by cyclin-dependent kinases.

Protein phosphatase 1 and protein phosphatase 2A contain potential phosphorylation sites for cyclin-dependent kinases. In the present study we found that rabbit skeletal muscle protein phosphatase 1, as well as recombinant protein phosphatase 1 alpha and protein phosphatase 1 gamma 1, but not protein phosphatase 2A, was phosphorylated and inhibited by cdc2/cyclin A and cdc2/cyclin B. Phosphopeptide mapping and phospho amino acid analysis suggested that the phosphorylation site was located at a C-terminal threonine. Neither cdc2/cyclin A nor cdc2/cyclin B phosphorylated an active form of protein phosphatase 1 alpha in which Thr-320 had been mutated to alanine, indicating that the phosphorylation occurred at this threonine residue. Furthermore, protein phosphatase 1, but not protein phosphatase 2A, activity was found to change during the cell cycle of human MG-63 osteosarcoma cells. The observed oscillations in protein phosphatase 1 activity during the cell cycle may be due, at least in part, to phosphorylation of protein phosphatase 1 by cyclin-dependent kinases. Together, the results suggest a mechanism for direct regulation of protein phosphatase 1 activity.     

A domain for editing by an archaebacterial tRNA synthetase

The rules of the genetic code are established by aminoacylations of transfer RNAs by aminoacyl tRNA synthetases. New codon assignments, and the introduction of new kinds of amino acids, are blocked by vigorous tRNA-dependent editing reactions occurring at hydrolytic sites embedded within specialized domains in the synthetases. For some synthetases, these domains were present at the time of the last common ancestor and were fixed in evolution through all three of the kingdoms of life. Significantly, a well characterized domain for editing found in bacterial and eukaryotic threonyl- and all alanyl-tRNA synthetases is missing from archaebacterial threonine enzymes. Here we show that the archaebacterial Methanosarcina mazei ThrRS efficiently misactivates serine, but does not fuse serine to tRNA. Consistent with this observation, the enzyme cleared serine that was linked to threonine-specific tRNAs. M. mazei and most other archaebacterial ThrRSs have a domain, N2(A), fused to the N terminus and not found in bacterial or eukaryotic orthologs. Mutations at conserved residues in this domain led to an inability to clear threonine-specific tRNA mischarged with serine. Thus, these results demonstrate a domain for editing that is distinct from all others, is restricted to just one branch of the tree of life, and was most likely added to archaebacterial ThrRSs after the eukaryote/archaebacteria split.     

Isolation of two interferon-induced translational inhibitors: a protein kinase and an oligo-isoadenylate synthetase.

Large-scale purification of translational inhibitors present in interferon-treated mouse L cells, but not in untreated cells, led to the isolation of two interferon-induced activities. One is a protein kinase system that is activatable by double-stranded RNA and ATP and that phosphorylates a Mr 67,000 protein and the smallest subunit of eukaryotic initiation factor-2. The purified protein kinase is a strong translational inhibitor. The second activity is an enzyme that, with double-stranded RNA, slowly polymerizes ATP into oligoadenylate with a 2'-5' phosphodiester linkage. The oligo-isoadenylate in turn activates a potent inhibitor of mRNA translation.     

Increased activity of cyclin-dependent kinase 5 leads to attenuation of cocaine-mediated dopamine signaling

Cocaine, a drug of abuse, increases synaptic dopamine levels in the striatum by blocking dopamine reuptake at axon terminals. Cyclin-dependent kinase 5 (Cdk5) and its activator p35, proteins involved in phosphorylation of substrates in postmitotic neurons, have been found to be up-regulated after chronic exposure to cocaine. To further examine the effects of Cdk5 and p35 induction on striatal dopamine signaling, we generated two independent transgenic mouse lines in which Cdk5 or p35 was overexpressed specifically in neurons. We report here that increased Cdk5 activity, as a result of p35 but not of Cdk5 overexpression, leads to attenuation of cocaine-mediated dopamine signaling. Increased Cdk5-mediated phosphorylation of dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa (DARPP-32) at Thr-75, was accompanied by decreased phosphorylation of DARPP-32 at Thr-34. Increased Cdk5-mediated phosphorylation of extracellular signal-regulated kinase kinase 1 at Thr-286 was accompanied by decreased activation of extracellular signal-regulated kinase 1/2. These effects contributed to attenuation of cocaine-induced phosphorylation of cAMP response element-binding protein as well as a lesser induction of c-fos in the striatum. These results support the idea that Cdk5 activity is involved in altered gene expression after chronic exposure to cocaine and hence impacts the long-lasting changes in neuronal function underlying cocaine addiction.     

Phosphorylation and activation of cAMP-dependent protein kinase by phosphoinositide-dependent protein kinase

Although phosphorylation of Thr-197 in the activation loop of the catalytic subunit of cAMP-dependent protein kinase (PKA) is an essential step for its proper biological function, the kinase responsible for this reaction in vivo has remained elusive. Using nonphosphorylated recombinant catalytic subunit as a substrate, we have shown that the phosphoinositide-dependent protein kinase, PDK1, expressed in 293 cells, phosphorylates and activates the catalytic subunit of PKA. The phosphorylation of PKA by PDK1 is rapid and is insensitive to PKI, the highly specific heat-stable protein kinase inhibitor. A mutant form of the catalytic subunit where Thr-197 was replaced with Asp was not a substrate for PDK1. In addition, phosphorylation of the catalytic subunit can be monitored immunochemically by using antibodies that recognize Thr-197 phosphorylated enzyme but not unphosphorylated enzyme or the Thr197Asp mutant. PDK1, or one of its homologs, is thus a likely candidate for the in vivo PKA kinase that phosphorylates Thr-197. This finding opens a new dimension in our thinking about this ubiquitous protein kinase and how it is regulated in the cell.     

蛛网膜下腔出血大鼠大脑皮质周期蛋白依赖激酶5的表达

目的 探讨蛛网膜下腔出血（subarachnoid hemorrhage,SAH）后大鼠大脑皮质细胞周期蛋白依赖性激酶5（cyclin-dependent kinase 5,Cdk5）的表达和细胞定位.方法 52只雄性Sprague-Dawley大鼠随机分为假手术组（n=12）和SAH组（n=40）,后者再随机分为SAH后6h、12 h、24 h、2d和3d组,每组8只.采用大鼠视交叉前池注血制备大鼠SAH模型.应用蛋白质印迹法、免疫组化法检测大鼠脑皮质Cdk5表达.双标记免疫荧光染色法检测Cdk5蛋白在大脑皮质的细胞定位,神经元核抗原标记神经元,胶质纤维酸性蛋白标记星形胶质细胞.结果 蛋白质印迹显示,SAH后12 h时大鼠脑皮质Cdk5蛋白表达上调（t=3.709,P=0.001）,1d时达高峰（t=3.475,P=0.002）.免疫组化显示,SAH后Cdk5阳性细胞比例也逐渐增高,且时程变化与蛋白质印迹结果一致,于1d时达高峰（=4.320,P=0.000）.双标记免疫荧光检测显示,假手术组Cdk5主要表达于神经元细胞质,而SAH组Cdk5向神经元细胞核中移位.Cdk5主要与星形胶质细胞和神经元之间存在共定位.结论 SAH可使大脑皮质Cdk5蛋白表达上调,Cdk5可能参与了SAH后早期脑损伤.     

Phosphorylation and inactivation of glycogen synthase kinase 3 by protein kinase A

Glycogen synthase kinase 3 (GSK-3) is implicated in multiple biological processes including metabolism, gene expression, cell fate determination, proliferation, and survival. GSK-3 activity is inhibited through phosphorylation of serine 21 in GSK-3 alpha and serine 9 in GSK-3 beta. These serine residues of GSK-3 have been previously identified as targets of protein kinase B (PKB/Akt), a serine/threonine kinase located downstream of phosphatidylinositol 3-kinase. Here, we show that serine 21 in GSK-3 alpha and serine 9 in GSK-3 beta are also physiological substrates of cAMP-dependent protein kinase A. Protein kinase A physically associates with, phosphorylates, and inactivates both isoforms of GSK-3. The results indicate that depending on the stimulatory context, the activity of GSK-3 can be modulated either by growth factors that work through the phosphatidylinositol 3-kinase-protein kinase B cascade or by hormonal stimulation of G protein-coupled receptors that link to changes in intracellular cAMP levels.     

Regulation of NMDA receptors by cyclin-dependent kinase-5.

Members of the N-methyl-d-aspartate (NMDA) class of glutamate receptors (NMDARs) are critical for development, synaptic transmission, learning and memory; they are targets of pathological disorders in the central nervous system. NMDARs are phosphorylated by both serine/threonine and tyrosine kinases. Here, we demonstrate that cyclin dependent kinase-5 (Cdk5) associates with and phosphorylates NR2A subunits at Ser-1232 in vitro and in intact cells. Moreover, we show that roscovitine, a selective Cdk5 inhibitor, blocks both long-term potentiation induction and NMDA-evoked currents in rat CA1 hippocampal neurons. These results suggest that Cdk5 plays a key role in synaptic transmission and plasticity through its up-regulation of NMDARs.     

RNA recognition by designed peptide fusion creates "artificial" tRNA synthetase

The genetic code was established through aminoacylations of RNA substrates that emerged as tRNAs. The 20 aminoacyl-tRNA synthetases (one for each amino acid) are ancient proteins, the active-site domain of which catalyzes formation of an aminoacyl adenylate that subsequently reacts with the 3' end of bound tRNA. Binding of tRNA depends on idiosyncratic (to the particular synthetase) domains and motifs that are fused to or inserted into the conserved active-site domain. Here we take the domain for synthesis of alanyl adenylate and fuse it to "artificial" peptide sequences (28 aa) that were shown previously to bind to the acceptor arm of tRNAAla. Certain fusions confer aminoacylation activity on tRNAAla and on hairpin microhelices modeled after its acceptor stem. Aminoacylation was sensitive to the presence of a specific G:U base pair known to be a major determinant of tRNAAla identity. Aminoacylation efficiency and specificity also depended on the specific peptide sequence. The results demonstrate that barriers to RNA-specific aminoacylations are low and can be achieved by relatively simple peptide fusions. They also suggest a paradigm for rationally designed specific aminoacylations based on peptide fusions.     

Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics

Pre-steady-state kinetic studies of Escherichia coli glutaminyl-tRNA synthetase conclusively demonstrate the existence of long-distance pathways of communication through the protein-RNA complex. Measurements of aminoacyl-tRNA synthesis reveal a rapid burst of product formation followed by a slower linear increase corresponding to k(cat). Thus, a step after chemistry but before regeneration of active enzyme is rate-limiting for synthesis of Gln-tRNA(Gln). Single-turnover kinetics validates these observations, confirming that the rate of the chemical step for tRNA aminoacylation (k(chem)) exceeds the steady-state rate by nearly 10-fold. The concentration dependence of the single-turnover reaction further reveals that the glutamine K(d) is significantly higher than the steady-state K(m) value. The separation of binding from catalytic events by transient kinetics now allows precise interpretation of how alterations in tRNA structure affect the aminoacylation reaction. Mutation of U35 in the tRNA anticodon loop decreases k(chem) by 30-fold and weakens glutamine binding affinity by 20-fold, demonstrating that the active-site configuration depends on enzyme-tRNA contacts some 40 A distant. By contrast, mutation of the adjacent G36 has very small effects on k(chem) and K(d) for glutamine. Together with x-ray crystallographic data, these findings allow a comparative evaluation of alternative long-range signaling pathways and lay the groundwork for systematic exploration of how induced-fit conformational transitions may control substrate selection in this model enzyme-RNA complex.