Purification, Cloning, and Characterization of Nek8, a Novel NIMA-related Kinase, and Its Candidate Substrate Bicd2

Abstract

We describe the isolation, cloning, and characterization of human Nek8, a new mammalian NIMA-related kinase, and its candidate substrate Bicd2. Nek8 was isolated as a ␤-casein kinase activity in rabbit lung and has an N-terminal catalytic domain homologous to the Nek family of protein kinases. Nek8 also contains a central domain with homology to RCC1, a guanine nucleotide exchange factor for the GTPase Ran, and a C-terminal coiled-coil domain. Like Nek2, Nek8 prefers ␤-casein over other exogenous substrates, has shared biochemical requirements for kinase activity, and is capable of autophosphorylation and oligomerization. Nek8 activity is not cell cycle regulated, but like Nek3, levels are consistently higher in G 0 -arrested cells. During the purification of Nek8 a second protein co-chromatographed with Nek8 activity. This protein, Bicd2, is a human homolog of the Drosophila protein Bicaudal D, a coiled-coil protein. Bicd2 is phosphorylated by Nek8 in vitro, and the endogenous proteins associate in vivo. Bicd2 localizes to cytoskeletal structures, and its subcellular localization is dependent on microtubule morphology. Treatment of cells with nocodazole leads to dramatic reorganization of Bicd2, and correlates with Nek8 phosphorylation. This may be indicative of a role for Nek8 and Bicd2 associated with cell cycle independent microtubule dynamics.Among the multiple families of related protein kinases that have been described, the mammalian NIMA related kinases, or Neks, have proven to be elusive in their functional characterization. For the most part, the relatedness of these kinases is restricted to sequence homology within the catalytic domain, as none of the Neks appear to be functionally related to NIMA.

Nercc1, a mammalian NIMA-family kinase, binds the Ran GTPase and regulates mitotic progression

“…It lacks the C-terminal tail that in Nercc1 spans from the polyglycine region to the end of the protein; this is an important regulatory region in Nercc1 (see below). A second protein kinase identical in sequence to Nercc1 has been deposited in GenBank; this can be accessed both as Nek8 (AY048580) and Nek9 (NM033116) (Holland et al 2002).…”

TINA Interacts with the NIMA Kinase inAspergillus nidulansand Negatively Regulates Astral Microtubules during Metaphase Arrest

“…NIMA-related kinases (NEKs) have also been identified in higher eukaryotes (Letwin et al, 1992;Schultz and Nigg, 1993;Lu and Hunter, 1995b;Chen et al, 1999;Tanaka and Nigg, 1999;Uto and Sagata, 2000;Kandli et al, 2000;Holland et al, 2002;Roig et al, 2002), some of which have been implicated in cell cycle progression.…”

Nek11, a New Member of the NIMA Family of Kinases, Involved in DNA Replication and Genotoxic Stress Responses

“…Nek6 and Nek7 are cytoplasmic proteins lacking the C-terminal regulatory domain (18) and function as putative regulators for p70 ribosomal S6 kinase (29). Recently Nek8/Nercc1 has been identified as a ␤-casein kinase or a Nek6-binding protein harboring an RCC1-like motif uniquely in the NIMA family (20,21). Nek8/Nercc1 associates with putative substrates Bicd2 and Ran GTPase and appears to have a role in microtubule dynamics (20,21).…”

“…Protein kinases structurally related to fungal NIMA have been isolated from various organisms. Nim-1 from Neurospora crassa (8), Fin1 from Schizosaccharomyces pombe (9), Kin3 from Saccharomyces cerevisiae (10), and at least seven proteins from mammals designated Nek (NIMA-related kinase): Nek1 (11), Nek2 (12), Nek3 (12,13), Nek4/STK2 (14 -16), Nek6 (17,18), Nek7 (18,19), and Nek8/Nercc1 (20,21) have been published. All of the NIMA-related kinases share considerable primary sequence similarity with NIMA over the N-terminal catalytic domain.…”