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. 2015 Nov 2:6:8771.
doi: 10.1038/ncomms9771.

Mechanistic basis of Nek7 activation through Nek9 binding and induced dimerization

Tamanna Haq  1   2 , Mark W Richards  1   2 , Selena G Burgess  1   2 , Pablo Gallego  3 , Sharon Yeoh  1   2 , Laura O'Regan  1   2 , David Reverter  3 , Joan Roig  4 , Andrew M Fry  1   2 , Richard Bayliss  1   2
Affiliations

Mechanistic basis of Nek7 activation through Nek9 binding and induced dimerization

Tamanna Haq et al. Nat Commun. .

Abstract

Mitotic spindle assembly requires the regulated activities of protein kinases such as Nek7 and Nek9. Nek7 is autoinhibited by the protrusion of Tyr97 into the active site and activated by the Nek9 non-catalytic C-terminal domain (CTD). CTD binding apparently releases autoinhibition because mutation of Tyr97 to phenylalanine increases Nek7 activity independently of Nek9. Here we find that self-association of the Nek9-CTD is needed for Nek7 activation. We map the minimal Nek7 binding region of Nek9 to residues 810-828. A crystal structure of Nek7(Y97F) bound to Nek9(810-828) reveals a binding site on the C-lobe of the Nek7 kinase domain. Nek7(Y97F) crystallizes as a back-to-back dimer between kinase domain N-lobes, in which the specific contacts within the interface are coupled to the conformation of residue 97. Hence, we propose that the Nek9-CTD activates Nek7 through promoting back-to-back dimerization that releases the autoinhibitory tyrosine residue, a mechanism conserved in unrelated kinase families.

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Figures

Figure 1
Figure 1. The crystal structure of Nek7 bound to Nek9.
(a) Cartoon representation of Nek7 (teal, chain A) and stick representation of Nek9 810–825 (orange chain C). (b) Cartoon representation of Nek7 (pink, chain B) and Nek9 810–818 (orange, chain D). (c) Surface representation of Nek7 (teal, chain A) and stick representation of Nek9 810–825 (chain C). Carbon atoms in Nek9 are colour-coded according the results of the mutagenesis/co-precipitation data (Supplementary Fig. 1b,c): key binding residues (orange), non-essential residues (yellow) and untested residues (wheat). (d) Helical wheel plot of the Nek9 α-helix with residues colour-coded by chemical properties (inner circle: basic, blue; acidic, red; bulky hydrophobic, black; other, white) and contribution to Nek7 binding (outer circle, coloured as carbon atoms in c).
Figure 2
Figure 2. Detailed interactions within the Nek7–Nek9 interface.
(a) Detailed view of the Nek7–Nek9 interaction in the region of Nek9 aa 810–816. (b) Detailed view of the Nek7–Nek9 interaction in the region of Nek9 aa 814–825. (c) Coomassie-stained SDS–PAGE gel showing the results of a GST co-precipitation experiment testing the binding of GST-Nek9-CTD to structure-based mutants of Nek7. (d) Mutagenesis/co-precipitation data from c mapped onto the surface of Nek7. Key binding residues (red), non-essential residues (green) and untested residues (teal).
Figure 3
Figure 3. Nek9 self-association stimulates Nek7 activation.
(a) In vitro kinase activity assay using a variety of Nek9 constructs to investigate the effect of Nek9 self-association on Nek7 autophosphorylation and substrate phosphorylation. The upper panel shows the Coomassie-stained gel (CB) and autoradiograph (32P). Incorporation of radioisotope was quantified by scintillation counting and is shown in the histogram (lower panel). Error bars represent the s.d. for two independent experiments. (b,c) Back-to-back dimerization of Nek7 is mediated through interactions between secondary structure elements, and the interface is centred around residue 97 (red).
Figure 4
Figure 4. Relationship between the conformation of residue 97 and the back-to-back interface.
(a,c,e) Active site viewed centred on residue 97. (b,d,f) View of the αC-β4 region that is involved in the back-to-back interface. In panels (d) and (f), the dimeric partner is shown as a thin ribbon with key residues shown as in sticks. The asterisk on Asn90* indicates that it belongs to the dimeric partner. Structures shown are: (a,d) Nek7WT alone (PDB code 2WQN, coloured grey); (b,e) Nek7Y97F, chain A (coloured teal); (c,f) Nek7 Y97F, chain B (coloured pink). (g) Schematic illustrations of the coupling between the conformation of residue 97 and the back-to-back interface.
Figure 5
Figure 5. Mutations in the Nek7 back-to-back interface.
(a) Structure of autoinhibited Nek7 showing residues involved in the back-to-back interface. (b) Structure of Nek7 in the back-to-back conformation. (c,d) In vitro kinase activity assay of WT Nek7 and Nek7 mutants alone and in the presence of Nek9-CTD. Kinase activity was quantified by scintillation counting. Error bars represent the standard error for two independent reactions. *P<0.05, **P<0.01 and ****P<0.0001 using one-way analysis of variance with Dunnett's post hoc test compared with the WT reaction for Nek7 alone reactions or to the WT plus N9 CTD reaction for assays performed in the presence of Nek9-CTD.
Figure 6
Figure 6. Biological relevance of Nek7 allosteric activation by Nek9.
(a,b) HeLa cells were transfected with RNAi resistant Flag-Nek7 WT or binding-deficient mutants as indicated, and grown in selection media containing G418 for 7 days to enrich the transfected cell population. Cells were then either mock- or Nek7-depleted for 72 h before being fixed and processed for immunofluorescence microscopy with Flag and phospho-H3 antibodies. The mitotic index of transfected cells was counted. Data represent mean (±s.d.) for three separate experiments where n=100–200 cells. Statistical analyses were carried out using a one-way analysis of variance followed by post hoc testing. *P<0.05, **P<0.01 and ***P<0.005.
Figure 7
Figure 7. Schematic model of Nek7 activation by Nek9 binding and induced dimerization.
The autoinhibited state of Nek7 (grey) is characterized by the Tyr-down conformation, in which the active site is blocked by a collapsed set of four R-spine residues (green). Autophosphorylation of Nek7 is slow and can be accelerated by mutation (Tyr97 and Lys96) or Nek9. Binding of self-associated Nek9 induces back-to-back dimerization of Nek7, which releases autoinhibition by promoting a Tyr-up conformation. Nek7 autophosphorylation results in an active kinase conformation. Once phosphorylated, Nek7 can remain bound to Nek9, but Nek9 is no longer required for Nek7 activity. Phosphorylation of Nek7 by Nek9 kinase activity is an alternative mechanism in vivo.
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