No difference in kinetics of tau or histone phosphorylation by CDK5/p25 versus CDK5/p35 in vitro

Abstract

CDK5/p35 is a cyclin-dependent kinase essential for normal neuron function. Proteolysis of the p35 subunit in vivo results in CDK5/p25 that causes neurotoxicity associated with a number of neurodegenerative diseases. Whereas the mechanism by which conversion of p35 to p25 leads to toxicity is unknown, there is common belief that CDK5/p25 is catalytically hyperactive compared to CDK5/p35. Here, we have compared the steady-state kinetic parameters of CDK5/p35 and CDK5/p25 towards both histone H1, the best known substrate for both enzymes, and the microtubule-associated protein, tau, a physiological substrate whose in vivo phosphorylation is relevant to Alzheimer's disease. We show that the kinetics of both enzymes are the same towards either substrate in vitro. Furthermore, both enzymes display virtually identical kinetics towards individual phosphorylation sites in tau monitored by NMR. We conclude that conversion of p35 to p25 does not alter the catalytic efficiency of the CDK5 catalytic subunit by using histone H1 or tau as substrates, and that neurotoxicity associated with CDK5/p25 is unlikely attributable to CDK5 hyperactivation, as measured in vitro.Alzheimer's disease | neurotoxicity | NMR | protein kinase | proteolysis C DK5/p25 was first identified as a tau kinase (1, 2) that displayed a CDK1 (formerly p34 cdc2 ) -like substrate specificity in brain (3, 4). Of particular interest, phosphorylation of normal tau by CDK5/p25 could partially recapitulate several characteristics inherent to PHF-tau associated with Alzheimer's disease (1, 2). The CDK5 catalytic subunit was homologous to other CDKs and displayed ubiquitous expression (5), but p25 was originally discovered as a unique protein expressed predominantly in neurons, and generated by proteolytic cleavage of a larger protein precursor, p35 (6-9). Whereas CDK5/p35 is associated with normal neuron development and function (10), endogenous cleavage of p35 to p25 is associated with neuronal cell death, neuropathology, and is implicated in the progression of Alzheimer's disease (11-18), Parkinson's disease (19), and ALS (20). CDK5/p25, but not CDK5/p35, is toxic when overexpressed in transformed cell lines or when generated by cleavage of endogenous p35 in neurons (11,21,22). The cellular mechanism by which cleavage causes toxicity is not known, but in theory could relate to one or more known key differences between these two enzyme forms, including differences in subcellular distribution (11, 23), stability of the protein (11, 24), and/or cellular substrate specificity (11,25).There is common belief that the catalytic activity of CDK5/p25 is significantly elevated in comparison to CDK5/p35, and therefore that p25 causes "hyperactivity" of CDK5 compared to p35, contributing to its toxicity (10). To rigorously demonstrate its hyperactivity, however, it is necessary to show that the catalytic parameters associated with steady-state phosphorylation of substrates are different between the two enzymes. Hashiguchi et al. (26) previously conducted such experiments,...

Cdk5 activity in the brain – multiple paths of regulation

“…Similarly, calpastatin, an endogenous inhibitor of calpains, is substantially decreased in AD (Nixon, 2003), which could lead to deregulation of Cdk5 through increased formation of p25 (Sato et al, 2011). Although a few in vitro studies have shown that the complex between Cdk5 and p25 displays a higher activity than that between Cdk5 and p35, other studies have reported comparable activities (Peterson et al, 2010). The relative catalytic activities of the Cdk5-p35 and Cdk5-p25 complexes in vivo are not known.…”

Spectroscopic Studies of GSK3β Phosphorylation of the Neuronal Tau Protein and Its Interaction with the N-terminal Domain of Apolipoprotein E

“…In vivo as well as in vitro, Tau becomes a more favorable substrate for GSK3␤ when it is prephosphorylated by a non-proline-dependent kinase (25,26) such as the cAMP-dependent protein kinase (PKA) or by another proline-dependent kinase such as the CDK5 cyclin-dependent kinase (27,28). PKA is able in vitro to phosphorylate Tau at the Ser-208, Ser-214, Ser-324, Ser-416, Ser-356, and Ser-409 sites, with a clear preference for the Ser-214 site (29,30), whereas CDK5 phosphorylates Tau at the Ser-235 site (28,31). Godemann et al (32) studied in vitro the GSK3␤ phosphorylation of Tau441 by twodimensional phosphopeptide mapping, immunoblotting with phosphorylation-sensitive antibodies, and phosphopeptide sequencing.…”

Cyclin-Dependent Kinase 5 – An Emerging Player in Parkinson’s Disease Pathophysiology

“…Since p25 generation has been associated with excessive Cdk5 activation and neuronal loss in neurodegenerative disease (Patrick et al 1999, Cruz andTsai 2004), it has been speculated that Cdk5-p25 may be catalytically more active than Cdk5-p35. Nonetheless, a recent study demonstrated that the activity of Cdk5 is comparable regardless of whether it is associated with p35 or p25 (Peterson et al 2010). These findings collectively suggest that the aberrant Cdk5 activity associated with p25 generation is likely due to prolonged activation of Cdk5, and not an elevated Cdk5 catalytic activity.…”