The Notch signaling inhibitor DAPT down-regulates cdk5 activity and modulates the distribution of neuronal cytoskeletal proteins.
Notch signaling is critical for the development of the nervous system. Cyclin-dependent kinase 5 (cdk5) is a neuronal kinase involved in neuronal development and phosphorylates a number of neuronal cytoskeletal proteins. To determine the relationship between Notch and cdk5 signaling, we tested the effects of the Notch inhibitor, N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester (DAPT) on cdk5 expression, activity and cytoskeletal protein distribution in the rat cortical neurons in primary cultures. Neurons treated with 10 microM DAPT showed attenuated cdk5 activity in spite of an up-regulation of cdk5 protein level, consistent with a phenomenon reported in the cdk5 transgenic mice. Immunoblot and immunofluorescence analyses showed an increased level of cdk5, but not p35. Phospho-tau and phospho-neurofilament showed a shift from axons to cell bodies in DAPT-treated cells. DAPT-induced attenuation of cdk5 activity was restored by over-expression of p35 indicating that it interacted with cdk5 and up-regulated nascent cdk5 activity. p35 over-expression also rescued DAPT-induced translocation of phospho-tau and phospho-neurofilament. Immunoprecipitation followed by immunoblotting demonstrated that DAPT does not disrupt cdk5 and p35 interaction. Moreover, DAPT up-regulated neurogenin that is negatively regulated by Notch, and down-regulated Hes1, a downstream target of Notch, suggesting that Notch signaling in the cortical neurons was disrupted. Semi-quantitative and quantitative RT-PCR analyses confirmed that DAPT up-regulated cdk5 expression at the transcriptional level. These results establish a link between Notch signaling and cdk5 expression regulating neuronal cytoskeletal protein dynamics.
Kanungo
Jyotshnabala
J
Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
Zheng
Ya-Li
YL
Amin
Niranjana D
ND
Pant
Harish C
HC
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D051360:Cyclin-Dependent Kinase 5 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000187:drug effects / Q000378:metabolism*; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D018398:Homeodomain Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D010770:Phosphotransferases / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D051880:Receptors, Notch / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D000072056:Transcription Factor HES-1; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics; D014280:Triglycerides / Q000494:pharmacology*; D005680:gamma-Aminobutyric Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D016875:tau Proteins / Q000378:metabolism
D051792:Basic Helix-Loop-Helix Proteins; D051360:Cyclin-Dependent Kinase 5; D003598:Cytoskeletal Proteins; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D010770:Phosphotransferases; D012333:RNA, Messenger; D051880:Receptors, Notch; D000072056:Transcription Factor HES-1; D014280:Triglycerides; D005680:gamma-Aminobutyric Acid; D016875:tau Proteins; C492235:Cdk5r1 protein, rat; C489735:Hes1 protein, rat; C510879:neurogenin-1, rat; C057077:1,2-dilinolenoyl-3-(4-aminobutyryl)propane-1,2,3-triol; C495907:Cdk5 protein, rat