Structure, function, and regulation of neuronal Cdc2-like protein kinase

doi:10.1016/0197-4580(95)00014-6

Neurobiology of Aging

Volume 16, Issue 3, May–June 1995, Pages 263-268

https://doi.org/10.1016/0197-4580(95)00014-6 Get rights and content

Abstract

We have identified and purified from bovine brain a novel protein kinase which catalyzes in vitro phosphorylation of neurofilament proteins NF-H and NF-M and tau proteins at sites implicating the enzyme in the regulation of neurocytoskeleton dynamics and in Alzheimer pathology. The protein kinase displays a phosphorylation site specificity similar or identical to the cell cycle regulatory kinase, cdc2 kinase. The purified kinase is a heterodimer of a cdc2-like catalytic subunit, called cdk5, and a 25 kDa regulatory subunit. The regulatory subunit is essential for kinase activity, and it is derived from a 35 kDa protein, p35 by proteolysis. Northern blot analysis of tissue distribution indicates that cdk5 is widely distributed but especially rich in brain, whereas p35 expression is only found in brain. The protein kinase is therefore termed neuronal cdc2-like kinase. The neuron-specificity of the enzyme appears to be conferred by the regulatory subunit. During cell division, cdc2 kinase is regulated by complex phosphorylation mechanisms involving a network of specific protein kinases. Some of these kinases or their homologs have been found in mammalian brains and they may be involved in the regulation of neuronal cdc2-like kinase.

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A novel 6-aminopurine scaffold bearing an N9-cis-cyclobutyl moiety was designed using structure-based molecular design based on two known CDK inhibitors, dinaciclib and Cmpd-27. A series of novel 6-aminopurine compounds was prepared for structure–activity relationship (SAR) studies of CDK2 and CDK5 inhibitors. Among the compounds synthesized, compound 8l displayed potent CDK2 and CDK5 inhibitory activities with low nanomolar ranges (IC₅₀ = 2.1 and 4.8 nM, respectively) and showed moderate cytotoxicity in HCT116 colon cancer and MCF7 breast cancer cell lines. Here, we report the synthesis and evaluation of novel 6-aminopurine derivatives and present molecular docking models of compound 81 with CDK2 and CDK5.
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It is a nontraditional CDK, because it has no established role as a cell-cycle regulator, though it is responsible for cell cycle arrest in neurons [68,69]. Its best known substrates are cytoskeletal proteins, as it catalyzes the phosphorylation of neurofilaments and Tau proteins, which direct neuronal polarity [70]. Lee et al. found elevated CDK5 kinase activity in human AD brains compared to controls, and thus it is an attractive target in AD research [71].
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Thr320 of PP1 is a proline-directed phosphorylation site recognized by cyclin-dependent kinases: Cdk1 and Cdk2 (3-6). Cdk1 and Cdk2 are not expressed significantly in adult brain, whereas Cdk5 shares a very similar substrate specificity with Cdk1 and Cdk2 (30) and is the major cyclin-dependent kinase in the neurons of mammalian brain (10-12). Previous studies showed that Egr-1 activates Cdk5 in neurons by increasing the expression of p35 subunit of Cdk5 (15, 18).
The transcription factor Egr-1 activates cyclin-dependent protein kinase 5 (Cdk5) during nerve growth factor (NGF)-induced differentiation of PC12 cells into neurons (Harada, T. Morooka, T., Ogawa, S., and Nishida, E. (2001) Nat. Cell Biol. 3, 453-459). The downstream target of Cdk5 in the Egr-1/Cdk5 pathway is not clear. In this study, we observed that phosphorylation of protein phosphatase 1 (PP1) on Thr³²⁰ is reduced in brain extracts from Egr-1^-/- mice, indicating that a kinase downstream of Egr-1 phosphorylates PP1. In HEK 293 cells co-transfected with PP1 and Cdk5, Cdk5 phosphorylates PP1. In vitro, Cdk5 purified from bovine brain phosphorylates bacterially expressed recombinant PP1. In NGF-treated PC12 cells, inhibition of Cdk5 by olomoucine or silencing Cdk5 expression by small interfering RNA strategy, suppresses PP1 phosphorylation. Silencing Cdk5 expression by small interfering RNA also blocks NGF-induced neurite outgrowth. Overexpression of PP1 (wild type) promotes NGF-induced differentiation of PC12 cells, whereas that of PP1 (T320A) has no effect. Our data indicate that PP1 is a downstream target of the NGF/Egr-1/Cdk5 pathway during NGF-induced differentiation of PC12 cells and suggest that PP1 phosphorylation promotes neuronal differentiation.
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Similar to the induction performed by HSV-1, VZV induces CDK4 and CDK2/cyclin E expression and activity in infected fibroblasts (53). Most interestingly, CDK1 is exclusively active in dividing cells such as those supporting a VZV-productive infectious cycle, whereas CDK5 is a prominent kinase in neurons supporting only a latent infection (42). In summary, these experiments demonstrated for the first time that IE63 is phosphorylated by CDK1 in Vero cells and CDK5 in neuronal cells.
During the first stage of Varicella-Zoster virus (VZV) infection, IE63 (immediate early 63 protein) is mostly expressed in the nucleus and also slightly in the cytoplasm, and during latency, IE63 localizes in the cytoplasm quite exclusively. Because phosphorylation is known to regulate various cellular mechanisms, we investigated the impact of phosphorylation by roscovitine-sensitive cyclin-dependent kinase (RSC) on the localization and functional properties of IE63. We demonstrated first that IE63 was phosphorylated on Ser-224 in vitro by CDK1 and CDK5 but not by CDK2, CDK7, or CDK9. Furthermore, by using roscovitine and CDK1 inhibitor III (CiIII), we showed that CDK1 phosphorylated IE63 on Ser-224 in vivo. By mutagenesis and the use of inhibitors, we demonstrated that phosphorylation on Ser-224 was important for the correct localization of the protein. Indeed, the substitution of these residues by alanine led to an exclusive nuclear localization of the protein, whereas mutations into glutamic acid did not modify its subcellular distribution. When transfected or VZV-infected cells were treated with roscovitine or CiIII, an exclusive nuclear localization of IE63 was also observed. By using a transfection assay, we also showed that phosphorylation on Ser-224 and Thr-222 was essential for the down-regulation of the basal activity of the VZV DNA polymerase gene promoter. Similarly, roscovitine and CiIII impaired these properties of the wild-type form of IE63. These observations clearly demonstrated the importance of CDK1-mediated IE63 phosphorylation for a correct distribution of IE63 between both cellular compartments and for its repressive activity toward the promoter tested.

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Structure, function, and regulation of neuronal Cdc2-like protein kinase

Abstract

Substrate specificity characterization of a cdc2-like protein kinase purified from bovine brain

J. Biol. Chem.

Mitogen activated protein (MAP kinase transforms tau protein into an Alzheimer-like state

EMBO J.

The MO 15 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclin-dependent kinases (CDKs) through phosphorylation of Thr161 and its homologues

EMBO J.

Molecular characterization of microtubule-associated proteins tau and MAP 2

Trends Neurosci.

Protein sequence and mass spectromatic analysis of tau in the Alzheimer's Disease brain

J. Biol. Chem.

Phosphorylation of neurofilament H subunit at the tail domain by cdc2 kinase dissociate the association to microtubules

J. Biol. Chem.

Weellike gene in human cells

Nature

Identification of the 23 kDa subunit of tau protein kinase II as a putative activator of cdk5 in bovine brain

FEBS Lett.

Tau protein kinase I converts normal tau protein into A-69-like component of paired helical filaments

J. Biol. Chem.

A68: A major subunit of paired helical filaments and derivatized forms of normal tau

Science

Purification and characterization of a novel proline-directed protein kinase from bovine brain

J. Biol. Chem.

A brain-specific activator of cyclin-dependent kinase 5

Nature

Brain prolinedirected protein kinase is a neurofilament kinase which displays high sequence homology to p34cdc2

J. Biol. Chem.

Phosphorylation-dependent epitopes of neurofilament antibodies on tau protein and relationship with Alzheimer tau

Phosphorylation of tau protein by purified p34cd 28 and a related protein kinase from neurofilament

J. Biol. Chem.

Human Weel kinase inhibits cell division by phosphorylating p34cdc2 exclusively on Tyr15

EMBO J.

Phosphorylation of tau protein by purified p34^cd 28 and a related protein kinase from neurofilament