MOD_NEK2_2

NIMA (Never in mitosis A) was initially identified in filamentous fungi Aspergillus nidulans as a serine/threonine kinase vital for entry into mitosis (Oakley,1983). Kinases with structural and functional homology to NIMA known as NIMA (Never In Mitosis gene A)-related Kinase (NEK) have been identified throughout eukaryotes with a significant expansion of the family in higher eukaryotes while a single NIMA homologue exists in yeast, 2, 4 and 11 NIMA-related kinases were identified in Drosophila, C. elegans, and mammals respectively. Of the 11 human NEKs, NEK2 is more closely related to fungal kinase and is the most studied paralogue. NEK2 is a cell cycle-regulated kinase with low activity in G1, increased in S and G2, and diminished after mitotic onset. At the G1/S transition, the vertebrate NEK2 expresses two splice variants, NEK2A and NEK2B. Only the NEK2A variant contains the binding site necessary for substrate recognition and a KEN box destruction motif in the C-terminal region which mediates its APC/C-dependent protein degradation (Hames,2002; Boekhout,2015).
NEK2 is localized to the centrosome during interphase and early mitosis where it phosphorylates many of its substrates at serine/threonine residues. Either inhibition of the catalytic activity or knockdown of its substrates like cNap1, rootletin inhibits centrosome separation, spindle assembly, and formation of multinucleated cells. In kinetochores, the knockdown of NEK2 causes the displacement of the centromeric protein Mad2 from kinetochores and impairs chromosome segregation (Moniz,2011). NEK2 phosphorylation of the spindle assembly checkpoint component, Hec1 (NDC80) regulates kinetochore-microtubule binding (Fry,2002). So, these studies indicate that NEK2 may coordinate cell division on multiple levels, and the deregulation of NEK2 causes centrosome abnormalities and aneuploidy. ß-catenin is a reported substrate (Bahmanyar,2008) but it does not seem to have an accessible Nek2 site. Studies have shown that NEK2 is associated with disease progression in non-Hodgkin lymphomas. Only a few substrate proteins have been identified for most of the NEKs except NEK2 which makes it difficult to obtain a definitive phosphorylation motif for this group. For NEK2, a phosphorylation motif pattern can be developed based on oriented peptide library screening (OPLS) (van de Kooij,2019; Johnson,2023; Alexander,2011) and compared to sites reported in the well-known cell cycle substrates.
In general, most of the NEKs in the human kinome have a preference for hydrophobic and basic residues with a core consensus motif, [FLMW][^P][^P]([ST])[^P] with additional specificity differences. The +1 position prefers hydrophobic residues and +2 prefers basic residues, notably Arg. NEK1/3/4/5/8/11 preferentially phosphorylate threonine residues, while NEK6/7 mainly target serine residues. NEK2 and 9 have nearly equal specificity towards serine and threonine. But NEK10 is a dual-specificity kinase, that can phosphorylate tyrosine as well as serine/threonine. The structural difference within the P+1 loop and the catalytic loop of the kinase domain renders specificity. As in the case of NEK10, the unique residues, isoleucine in the APE-4 position, and threonine in the HRD+2 position makes the P+1 loop and catalytic loop of NEK 10 distinct from other kinases and enable it to phosphorylate both tyrosine and serine (van de Kooij,2019).
The specificity-determining binding residues of NEK2 are conserved among eukaryotes. The specificity for Phe/Trp and other hydrophobic residues at the -3 position from the phosphosite arises from a flat hydrophobic surface formed by two alanines, Ala95 and Ala145. Small residues at this position are found in all NEK2 homologues. Selection for Arg in the Ser/Thr-2 position arises from an electrostatic interaction with Glu208. The hydrophobic pocket formed by Phe176 in the activation loop and Pro180 and Met183 of the active site specifically recognize Ile and other hydrophobic amino acids in the Ser/Thr+1 position.
The canonical NEK2 motif is represented by [FLM][^P][^P]([ST])[^DEP] [HRK]. Currently, two versions of the phosphorylation motif are provided in ELM, a strong pattern and a more tolerant one. Due to the potential for false positive assignments, it is especially important that matches to the tolerant pattern are clearly conserved in orthologous Nek2 substrates.