Purification, cloning, and characterization of Nek8, a novel NIMA-related kinase, and its candidate substrate Bicd2.
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 beta-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 beta-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.
Holland
Pamela M
PM
Immunex Corporation, Seattle, Washington 98101, USA. hollandp@immunex.com
Milne
Alison
A
Garka
Kirsten
K
Johnson
Richard S
RS
Willis
Cynthia
C
Sims
John E
JE
Rauch
Charles T
CT
Bird
Timothy A
TA
Virca
G Duke
GD
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D004330:Drosophila / Q000201:enzymology; D005434:Flow Cytometry; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000201:enzymology; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008869:Microtubule-Associated Proteins; D008969:Molecular Sequence Data; D000072380:NIMA-Related Kinase 1; D000072379:NIMA-Related Kinases; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
C435206:Bicd2 protein, mouse; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; C062521:RCC1 protein, human; D011494:Protein Kinases; C000606914:NEK1 protein, human; C458529:NEK8 protein, human; C462119:NEK9 protein, human; D000072380:NIMA-Related Kinase 1; D000072379:NIMA-Related Kinases; C000606920:Nek1 protein, mouse; D017346:Protein Serine-Threonine Kinases