Protocols for Characterization of Cdk5 Kinase Activity

Abstract

Cyclin-dependent kinases (Cdks) are generally known to be involved in controlling the cell cycle, but Cdk5 is a unique member of this protein family for being most active in post-mitotic neurons. Cdk5 is developmentally important in regulating neuronal migration, neurite outgrowth, and axon guidance. Cdk5 is enriched in synaptic membranes and is known to modulate synaptic activity. Postnatally, Cdk5 can also affect neuronal processes such as dopaminergic signaling and pain sensitivity. Dysregulated Cdk5, in contrast, has been linked to neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Despite primarily being implicated in neuronal development and activity, Cdk5 has lately been linked to non-neuronal functions including cancer cell growth, immune responses, and diabetes. Since Cdk5 activity is tightly regulated, a method for measuring its kinase activity is needed to fully understand the precise role of Cdk5 in developmental and disease processes. This article includes methods for detecting Cdk5 kinase activity in cultured cells or tissues, identifying new substrates, and screening for new kinase inhibitors. Furthermore, since Cdk5 shares homology and substrate specificity with Cdk1 and Cdk2, the Cdk5 kinase assay can be used, with modification, to measure the activity of other Cdks as well.

Activation of Cyclin-Dependent Kinase 5 Broadens Action Potentials in Human Sensory Neurons

“…Two days after the transfection, cells were treated with various inflammatory mediators. CDK5 activity was measured as previously described 34 .…”

“…31.543017 doi: bioRxiv preprint 3 involved in pain signaling and then can lower their threshold of activation, affect channel desensitization, or enhance channel trafficking to the plasma membrane. Of these protein kinases, Cdk5 is unusual in that it shares a high degree of homology with other Cdk's that predominately regulates the cell cycle instead of being active in post-mitotic neurons 34 . In addition, Cdk5 activity is neither induced by a cyclin, as typical with other Cdk family members, nor with a second message molecule as seen with PKA, PKC, and the Ca 2+ /calmodulindependent protein kinase CaMKII.…”

“…Further studies have shown that inflammatory cytokines including tumor necrosis factor α, transforming growth factor β, and nerve growth factor can trigger ERK1/2 activation to subsequently boost p35 expression and ultimately lead to increased Cdk5 activity 34,37,38 .…”

TFP5, a Peptide Derived From Cdk5 Activator p35, Protects Pancreatic β-Cells From Glucose Toxicity

“…Kinase assays were performed as previously described 21 . Cdk5 was immunoprecipitated overnight from the supernatants of lysed cells using the polyclonal C-8 antibody at 4°C, and immunoglobulin isolation was performed with protein A-Sepharose beads for 2h at 4°C.…”

Activation of cyclin-dependent kinase 5 broadens action potentials in human sensory neurons

“…6 Further studies have shown that inflammatory cytokines including tumor necrosis factor α, transforming growth factor β, and nerve growth factor can trigger ERK1/2 activation to subsequently boost p35 expression and ultimately lead to increased Cdk5 activity. 4,7,8 Cdk5 hyperactivity in primary afferent neurons has consequently been implicated with pain hypersensitivity. The transgenic overexpression of p35 alone can, in effect, promote both thermal and mechanical pain in mice.…”