Sumoylation of p35 Modulates p35/Cyclin-Dependent Kinase (Cdk) 5 Complex Activity
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Cyclin-dependent kinase (Cdk) 5 is critical for central nervous system development and neuron-specific functions including neurite outgrowth as well as synaptic function and plasticity. Cdk5 activity requires association with one of the two regulatory subunits, called p35 and p39. p35 redistribution as well as misregulation of Cdk5 activity is followed by cell death in several models of neurodegeneration. Posttranslational protein modification by small ubiquitin-related modifier (SUMO) proteins (sumoylation) has emerged as key regulator of protein targeting and protein/protein interaction. Under cell-free in vitro conditions, we found p35 covalently modified by SUMO1. Using both biochemical and FRET-/FLIM-based approaches, we demonstrated that SUMO2 is robustly conjugated to p35 in cells and identified the two major SUMO acceptor lysines in p35, K246 and K290. Furthermore, different degrees of oxidative stress resulted in differential p35 sumoylation, linking oxidative stress that is encountered in neurodegenerative diseases to the altered activity of Cdk5. Functionally, sumoylation of p35 increased the activity of the p35/Cdk5 complex. We thus identified a novel neuronal SUMO target and show that sumoylation is a likely candidate mechanism for the rapid modulation of p35/Cdk5 activity in physiological situations as well as in disease.
KeywordsSumoylation p35/Cdk5 activity FRET/FLIM Oxidative stress
We thank Christine Poser and Claudia Fokken for excellent technical support. We thank Ron Hay (University of Dundee, Dundee, UK) for providing us with the His6-SUMO1 and His6-SUMO2 plasmids, Frauke Melchior (ZMBH, Heidelberg, Germany) for the components used for the in vitro sumoylation assay and Gertrude Bunt (University Medical Center Göttingen, Göttingen, Germany) for mVenus and mTFP plasmids. This work was supported by the Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB).
Conflict of Interest
The authors declare that they have no conflict of interest.
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