Abstract
Cyclin-dependent kinase 5 (CDK5) is a unique CDK, the activity of which can be detected in postmitotic neurons. To date, CDK5 purified from mammalian brains has always been associated with a truncated form of the 35-kDa major brain specific activator (p35, also known as nck5a) of CDK5, known as p25. In this study, we report that p35 can be cleaved to p25 both in vitro and in vivo by calpain. In a rat brain extract, p35 was cleaved to p25 by incubation with Ca(2+). This cleavage was inhibited by a calpain inhibitor peptide derived from calpastatin and was ablated by separating the p35.CDK5 from calpain by centrifugation. The p35 recovered in the pellet after centrifugation could then be cleaved to p25 by purified calpain. Cleavage of p35 was also induced in primary cultured neurons by treatment with a Ca(2+) ionophore and Ca(2+) and inhibited by calpain inhibitor I. The cleavage changed the solubility of the CDK5 active complex from the particulate fraction to the soluble fraction but did not affect the histone H1 kinase activity. Increased cleavage was detected in cultured neurons undergoing cell death, suggesting a role of the cleavage in neuronal cell death.
Highlights
Cyclin-dependent kinases (CDKs)1 are a group of serine/ threonine protein kinases activated by binding to a regulatory subunit cyclin
This is supported by the following observations: (i) Cyclin-dependent kinase 5 (CDK5) was purified from microtubule-enriched fractions of bovine and porcine brain extracts [23, 28]; (ii) CDK5 formed a multimolecular complex (ϳ670 kDa) that was eluted at void volume fractions from a gel filtration column [31]; (iii) using the yeast twohybrid system, p35 bound to neurofilaments and ␣-actinin [20, 32]; and (iv) immunofluorescent staining of cultured neurons suggested an association of p35 with actin filaments in growth cone [20]
Cleavage of p35 to p25 during Column Chromatography— After purification from bovine or porcine brains, CDK5 has been shown to consist of a 32-kDa catalytic subunit and a 23–26-kDa regulatory subunit, known as p25 [23, 28, 33]. p25 is the proteolytic C-terminal fragment of p35 [9, 10, 12]
Summary
Cyclin-dependent kinases (CDKs)1 are a group of serine/ threonine protein kinases activated by binding to a regulatory subunit cyclin. CDK5 Kinase Activity Assay—The brain (350 g), crude (50 g), or cultured neuron (50 g) extracts were prepared in 100 l of 20 mM MOPS, pH 7.2, containing 0.3 M NaCl, 1 mM MgCl2, 1 mM EGTA, 0.1 mM EDTA, 0.5% Nonidet P-40, 1 g/ml leupeptin, and 1 mM dithiothreitol. All p35 was converted to p25 after fractionation p35 CDK5 Activator Cleavage to p25 by Calpain through the hydroxyapatite column (Fig. 1, lane 2).
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