The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Functional site class:
NEK2 phosphorylation site
Functional site description:
NEK2 is a serine/threonine kinase that belongs to the Nek family of protein kinases. Homologous NEKs have been identified in many eukaryotes and are involved in a myriad of functions such as cilia regulation, microtubule dynamics, mitotic processes, cell growth, centrosome splitting, meiosis, and DNA damage response. NEK2 is the closest vertebrate homologue of the originator, NIMA of Aspergillus nidulans. It is a core component of the human centrosome and its expression exhibits a cell cycle-dependent pattern. NEK2 activity peaks in S and G2/M stages of cell cycle where it phosphorylates several centrosomal linker proteins such as C-Nap1, rootletin, LRRC45, Cep68 and leads to centrosome disjunction. NEK2 is overexpressed in a wide variety of cancers and contributes to increased drug resistance making it a target for intervention in cancer.
ELMs with same func. site: MOD_NEK2_1  MOD_NEK2_2 
ELM Description:
This is the tolerant version of the NEK2 site motif preferences. Since very few of the experimentally reported p-sites match the preferences identified by systematic peptide phosphorylation experiments, they could not be used for defining the specificity of NEK2. However, two oriented peptide library phosphorylation arrays (Alexander,2011; Johnson,2023) provide information on the NEK2 p-site preferences. Both hydrophobic and positive residues, particularly Arginine, are preferred in several positions. The arrays disfavour negative residues in most positions. Proline is also strongly disfavoured at several positions.

The strongest selectivity for NEK2 is found in the S/T-3 and S/T+2 positions. In the S/T-3 position, a subset of large hydrophobic residues Phe, Leu and Met have the strongest preferences. At S/T+2, Arg is the main preference, with His and Lys also favoured. The S/T+1 position is less strong but has a clear hydrophobic, mainly aliphatic preference, Met, Leu, Ile, Val. This position also disallows Pro, Asp, Glu, and also clearly disfavours Gly, Ala, Asn. Positions S/T-2 and -1 have the weakest preferences with R and K being the most preferred residues. Pro is the least preferred with Glu and Asp plus beta-branched Val and Ile residues being clearly disfavoured. Proline is strongly disfavoured in the Ser/Thr-2, Ser/Thr-1, and S/T+1 positions. Asp and Glu are also disfavoured in the S/T+1 position. The S/T+2 position shows a strong selection for Arg and His and disfavours Glu. In ELM, there are two versions of the motif pattern with stricter (MOD_NEK2_1) and more tolerant (MOD_NEK2_2) regular expressions based on the array data. Matches to the strict pattern should have more confidence than matches to the tolerant pattern. Conservation analysis is always important but is especially strongly advised for the weaker motif candidates.
Pattern: [FLMW][^P][^P]([ST])[^PDEGAN][RKH].
Pattern Probability: 0.0013894
Present in taxon: Metazoa
Interaction Domain:
Pkinase (PF00069) Protein kinase domain (Stochiometry: 1 : 1)
o Abstract
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.
o 18 selected references:

o 23 GO-Terms:

o No Instance for MOD_NEK2_2
Please cite: The Eukaryotic Linear Motif resource: 2022 release. (PMID:34718738)

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