Phosphoproteome and transcriptome analysis of the neuronal response to a CDK5 inhibitor

被引:22
作者
Gillardon, F
Steinlein, P
Bürger, E
Hildebrandt, T
Gerner, C
机构
[1] Boehringer Ingelheim Pharma GmbH & Co KG, CNS Res, D-88397 Biberach, Germany
[2] Res Inst Mol Pathol, A-1030 Vienna, Austria
[3] Univ Vienna, Canc Res Inst, Vienna, Austria
关键词
phosphoproteome; neurodegeneration; protein kinase; signal transduction; two-dimensional gel electrophoresis;
D O I
10.1002/pmic.200400992
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
In Alzheimer's disease and amyotrophic lateral sclerosis deregulation of cyclin-dependent kinase 5 (CDK5) causes hyperphosphorylation of tau and neurofilament proteins, respectively, leading to neuronal cell death. We have demonstrated recently that pharmacological inhibition of CDK5 protects neurons under various stressful conditions (Weishaupt J. H., et al., Molec. Cell. Neurosci. 2003, 24, 489-502). To get an overview on the cellular mechanisms of action we analyzed global changes in protein phosphorylation in cultured cerebellar granule neurons by [P-32]orthophosphate labeling after administration of a CDK5 inhibitor. Since CDK5 has recently been shown to phosphorylate and inactivate transcription factor MEF2, we included gene expression profiling using cDNA microarrays. By two-dimensional gel electrophoresis and matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF)-mass spectrometry we identified several phosphoproteins that were modulated by compound administration. Among them syndapin I which is involved in vesicle recycling, and dynein light intermediate chain 2 which represents a regulatory subunit of the dynein protein complex. These findings are consistent with the known physiological function of CDK5 in synaptic signaling and axonal transport. Moreover, we detected phosphoproteins acting in neuronal surival and/or neurite outgrowth, such as cofilin and collapsin response mediator protein. Subsequent testing in cell cultures revealed that the CDK5 inhibitor blocked mitochondrial translocation of pro-apoptotic cofilin in cerebellar granule neurons and enhanced neurite outgrowth in dorsal root ganglia. Numerous genes exhibiting MEF2 consensus binding sequences were modulated by CDK5 inhibitor treatment. Among them some that may contribute to neurite elongation or neuronal survival, but also several genes functioning in synaptic transmission. Taken together, phosphoproteome and transcriptome analysis indicate that the compound promotes both neuronal survival and neurite outgrowth, but also may affect synaptic function in cultured neurons.
引用
收藏
页码:1299 / 1307
页数:9
相关论文
共 64 条
[1]   Phosphorylation by cdc2-cyclinB1 kinase releases cytoplasmic dynein from membranes [J].
Addinall, SG ;
Mayr, PS ;
Doyle, S ;
Sheehan, JK ;
Woodman, PG ;
Allan, VJ .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2001, 276 (19) :15939-15944
[2]   Neuronal Cdc2-like protein kinase (Cdk5/p25) is associated with protein phosphatase 1 and phosphorylates inhibitor-2 [J].
Agarwal-Mawal, A ;
Paudel, HK .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2001, 276 (26) :23712-23718
[3]   Syntrophins and dystrobrevins: Defining the dystrophin scaffold at synapses [J].
Albrecht, DE ;
Froehner, SC .
NEUROSIGNALS, 2002, 11 (03) :123-129
[4]  
Aletta JM, 1996, J NEUROBIOL, V31, P461, DOI 10.1002/(SICI)1097-4695(199612)31:4<461::AID-NEU6>3.0.CO
[5]  
2-7
[6]   A rapid method for the isolation of metabolically active mitochondria from rat neurons and astrocytes in primary culture [J].
Almeida, A ;
Medina, JM .
BRAIN RESEARCH PROTOCOLS, 1998, 2 (03) :209-214
[7]   DETERMINATION OF THE CONSENSUS BINDING-SITE FOR MEF2 EXPRESSED IN MUSCLE AND BRAIN REVEALS TISSUE-SPECIFIC SEQUENCE CONSTRAINTS [J].
ANDRES, V ;
CERVERA, M ;
MAHDAVI, V .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (40) :23246-23249
[8]   Proteins of the ADF/cofilin family: Essential regulators of actin dynamics [J].
Bamburg, JR .
ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY, 1999, 15 :185-230
[9]   BIOCHEMICAL AND FUNCTIONAL DIVERSITY OF MICROTUBULE MOTORS IN THE NERVOUS-SYSTEM [J].
BRADY, ST ;
SPERRY, AO .
CURRENT OPINION IN NEUROBIOLOGY, 1995, 5 (05) :551-558
[10]  
Byk T, 1996, J NEUROSCI, V16, P688