Identification and Sequence Analysis of Six New Members of the NIMA‐related Kinase Family in Chlamydomonas

Abstract

The NIMA kinases are an evolutionarily conserved protein family with enigmatic roles in the regulation of mitosis. We report six new members of this family in Chlamydomonas, in addition to the previously identified NIMA-related kinase, Fa2p. Chlamydomonas NIMA-related kinases (CNKs) 1-6 were sequenced from subclones generated by RT-PCR using information from EST libraries and the recently sequenced Chlamydomonas genome. Phylogenetic and bioinformatic approaches were used to determine the relationships of the six new members with known members of the NIMA-related kinase family. Although humans express at least eleven NIMA-related kinases, the eukaryotic microbes that have been studied to date express only one or two members of the family. Thus, the discovery that Chlamydomonas expresses a total of at least seven NIMA-related kinases is intriguing. Our analyses suggest that members of this family may play roles in the assembly and function of cilia.

A NIMA-related kinase, Cnk2p, regulates both flagellar length and cell size inChlamydomonas

“…The conservation of Fa2p localization suggests that one or more mammalian NIMA-related kinases may be axonemal. We have previously noted that expansion of the NIMA-related family of proteins correlates with the presence of ciliated cells in the lineage (Bradley et al, 2004). Of the ten known NIMA-related kinase genes in Chlamydomonas, only two have been studied to date (FA2 and CNK2) and both affect ciliary disassembly and the cell cycle.…”

“…The ble coding sequence (start codon to stop codon) was removed from pGenD by digestion with NdeI and EcoRI (New England Biolabs, Beverly, MA; Invitrogen, Burlington, ON) to be replaced by the CNK2 coding sequence. The CNK2 coding sequence was amplified with Pfu Turbo polymerase (Stratagene, La Jolla, CA) from the cloned cDNA (Bradley et al, 2004) using primers designed to include NdeI and EcoRI restriction sites at the start and just before the stop codons, respectively (forward, 5′-CGATCATATGGCGAAGGACGGGTCT-AAGG-3′ and reverse, 5′-CGATGAATTCCCACCCGCGCGCTGG-CCGCGG-3′). The product was digested with NdeI and EcoRI, and ligated using T4 DNA ligase (Invitrogen) to the pGenD backbone.…”

“…Eukaryotes that lack cilia, such as fungi and higher plants, only have one or a few members of this family (Osmani et al, 1991;Kambouris et al, 1993;Krien et al, 1998). Organisms with ciliated cells, such as Chlamydomonas and humans, have ten or eleven members (reviewed by O'Connell et al, 2003;Bradley et al, 2004) (our unpublished observations). This, in conjunction with the association Fa2p with cilia, raises the possibility that this gene family has ciliarelated functions in addition to established cell cycle roles (reviewed by O'Connell et al, 2003).…”

Members of the NIMA-related Kinase Family Promote Disassembly of Cilia by Multiple Mechanisms

“…Two additional well supported subfamilies do not have animal homologues, but they include NRKs of Chlamydomonas (Bradley et al, 2004) and Tetrahymena and may be protist specific. The FA2 subfamily includes Fa2p and Cnk4p NRKs of Chlamydomonas and Tetrahymena Nrk8p, Nrk11p, and Nrk17p.…”

“…Nrk18p and Nrk20p are homologues of the mammalian Nek2, implicated in the splitting of duplicated centrosomes (Fry et al, 1998;Faragher and Fry, 2003). The second conserved subfamily includes human Nek6 and Nek7, which have been implicated in the metaphase-anaphase transition (Yin et al, 2003), and a single Tetrahymena homologue, Nrk32p.Two additional well supported subfamilies do not have animal homologues, but they include NRKs of Chlamydomonas (Bradley et al, 2004) and Tetrahymena and may be protist specific. The FA2 subfamily includes Fa2p and Cnk4p NRKs of Chlamydomonas and Tetrahymena Nrk8p, Nrk11p, and Nrk17p.…”