Ndr/Lats Kinases Bind Specific Mob-Family Coactivators through a Conserved and Modular Interface

Abstract

Ndr/Lats kinases bind Mob coactivator proteins to form complexes that are essential and evolutionarily conserved components of "Hippo" signaling pathways, which control cell proliferation and morphogenesis in eukaryotes. All Ndr/Lats kinases have a characteristic N-terminal regulatory (NTR) region that binds a specific Mob cofactor: Lats kinases associate with Mob1 proteins, and Ndr kinases associate with Mob2 proteins. To better understand the significance of the association of Mob protein with Ndr/Lats kinases and selective binding of Ndr and Lats to distinct Mob cofactors, we determined crystal structures of Saccharomyces cerevisiae Cbk1^NTR-Mob2 and Dbf2^NTR-Mob1 and experimentally assessed determinants of Mob cofactor binding and specificity. This allowed a significant improvement in the previously determined structure of Cbk1 kinase bound to Mob2, presently the only crystallographic model of a full length Ndr/Lats kinase complexed with a Mob cofactor. Our analysis indicates that the Ndr/Lats^NTR-Mob interface provides a distinctive kinase regulation mechanism, in which the Mob cofactor organizes the Ndr/Lats NTR to interact with the AGC kinase C-terminal hydrophobic motif (HM), which is involved in allosteric regulation. The Mob-organized NTR appears to mediate association of the HM with an allosteric site on the N-terminal kinase lobe. We also found that Cbk1 and Dbf2 associated specifically with Mob2 and Mob1, respectively. Alteration of residues in the Cbk1 NTR allows association of the noncognate Mob cofactor, indicating that cofactor specificity is restricted by discrete sites rather than being broadly distributed. Overall, our analysis provides a new picture of the functional role of Mob association and indicates that the Ndr/Lats^NTR-Mob interface is largely a common structural platform that mediates kinase-cofactor binding.

Figure 1.

Crystal structure of the Cbk1^NTR–Mob2 complex that highlights conserved structural motifs at the Ndr/Lats–Mob interface. (A) Cbk1^NTR (residues 251–351, blue) complexed with zinc-binding Mob2 (residues 45–287, orange). (B) Cbk1^NTR–Mob2 (blue/orange) overlaid with Lats1^NTR–hMob1 (PDB entry 5B5W, cyan/purple). (C) Alignment of the Ndr/Lats NTR regions across eukaryotes. Abbreviations: Ce, Caenorhabditis elegans; Dm/Wts, Drosophila melanogaster; Xl, Xenopus laevis; Co, Capsaspora owczarzaki. Basic residues are colored blue, acidic residues red, and hydrophobic regions yellow. Interfaces I–III equivalent to those described in the Lats1–hMob1 structure are labeled. Conserved residues shown in Cbk1 are labeled. (D) Cbk1^NTR–Mob2 (top) and Lats1^NTR–Mob1 interaction architecture. The NTR is comprised of two Mob-binding helices: αMobA and αMobB. Conserved residues are labeled in bold.

Figure 2.

Dbf2 interacts with Mob1 through conserved residues. (A) Crystal structure of Dbf2^NTR–Mob1 with conserved motifs labeled: Dbf2, green; Mob1, dark red. (B) Interaction architecture of interacting residues within the Dbf2 NTR helices αMobA and αMobB (left) and between Dbf2 NTR and Mob1 (right). Conserved residues are labeled in bold. (C) MBP-fused Dbf2 NTR (residues 85–173) and His6-fused Mob1 (residues 79–314) co-expressed in E. coli followed by co-purification of the cognate binding proteins with nickel resin. Samples were subjected to SDS–PAGE and stained with Coomassie. The bottom panel represents Western blots of the lysate as a loading control. For quantification (right), the MBP-Dbf2 NTR Coomassie signal was normalized to the input anti-MBP signal and divided by the bait Mob1 Coomassie signal. The resulting ratios were normalized to the wild-type input (WT) and averaged over four replicates (n = 4). Error bars represent the standard deviation: ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; or ****p < 0.0001, based on a two-tailed t test (n = 4).

Figure 3.

Conserved regions cohere Ndr/Lats^NTR–Mob complexes. (A) Comparison of interface II from Lats1^NTR–hMob1 (PDB entry 5B5W, center panel) with Cbk1^NTR–Mob2 (right) and Dbf2^NTR–Mob1 (left). Highlighted residues shown as sticks are important for the interaction. (B) Comparisons of interface III from Lats1^NTR–hMob1 (center), Cbk1^NTR–Mob2 (right), and Dbf2^NTR–Mob1 (left). (C) More detailed view of interface II of the Cbk1^NTR–Mob2 complex showing the orientation of Cbk1 Arg-304 in relation to Leu-340 and Mob2 Val-120 (left) and Cbk1 Glu-312 and Mob2 Lys-118 and Tyr-119 (right). The interaction between Lys-118/Tyr-119 of Mob2 and Glu-312/Leu-329 of Cbk1 is distinct from those of Lats and Dbf2–Mob1 complexes. (D) Generalized architecture of Dbf2/Lats1–Mob1 in relation to that of Cbk1–Mob2. Conserved binding interfaces II and III as shown in panels A–C are boxed. Basic residues are colored blue, and acidic residues red. Mob is colored black, and the Ndr/Lats kinase NTR is colored gray.

Figure 4.

Ndr/Lats–Mob binding is specific and controlled through a three-residue motif. (A) Mob–NTR interaction specificity is maintained in vivo. Yeast two-hybrid test of kinase–NTR complexes. Selection: yeast cells containing binding domain (BD)–Mob and activation domain (AD)–NTR fragments on dropout agar serially diluted. Growth on this “Selection” agar plate is indicative of binding between BD and AD constructs (BD, DNA-binding domain construct; AD, transcriptional activation domain construct; −, empty plasmid). Viability/growth “Control” plate: single AD construct transformants grown on nonselective YPD agar plates. (B) Dbf2 and Cbk1 form specific complexes with Mob1 and Mob2, respectively, in vitro. MBP-fused Dbf2 NTR (85–173), labeled as “D”, and Cbk1^NTR (251–351), labeled as “C”, were co-expressed with His6-fused Mob1 and Mob2 V148C Y153C in E. coli, isolated via nickel chromatography, separated via SDS–PAGE, and stained with Coomassie. The dotted line demarcates a division in the gel. Bottom panels show Western blots of the E. coli lysate using anti-MBP antibody (αMBP). NTR, intact MBP–NTR fusion. (C) Quantification of panel B. The Coomassie signal from prey Dbf2–Cbk1 was normalized to the amount in the total lysate and divided by the total bait signal. Three individual replicates were performed. Samples were normalized to the “cognate” Dbf2–Mob1 (green) or Cbk1–Mob2 (orange) signals [ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; or ****p < 0.0001, based on a two-tailed t test (n = 3)]. (D) Diagram of wild-type (WT) and “swapped” Mob cofactors. The kinase restrictor motif (KRM), a short, three-amino acid motif at the interaction interface (purple) between Mob and the kinase NTR, was mutated to reflect the paralogous sequence in Mob1 and Mob2. Mob1^SWAP was mutated to possess the Mob2 sequence (KYV); Mob2^SWAP has the KRM mutated to the Mob1 sequence (RGE). (E and F) Mob1^SWAP and Mob2^SWAP mutants gain the ability to bind noncognate kinases. in the top panels, wild-type (WT) or His6-fused swapped (SWAP) Mob1 (E) or Mob2 (F) was co-expressed with MBP-fused Dbf2 or Cbk1 NTR in E. coli, isolated via nickel chromatography, separated via SDS–PAGE, and stained with Coomassie. The middle panels show the quantification of the top panels as in panel B. The bottom panels show Western blots of the E. coli lysate using the anti-MBP antibody (αMBP). Kinase^NTR, intact MBP–NTR fusion.

Figure 5.

Revised Cbk1–Mob2 crystal structure that reveals Ndr structural components conserved in Ndr1/Lats1. (A) The crystal structure of the inactive Cbk1–Mob2 complex was remodeled on the basis of the new Cbk1^NTR–Mob2 structure. In the new model, the coactivator binds to two N-terminal helices (αMobA and αMobB) that together with the top of the kinase domain (β3) form a binding slot for the AGC kinase hydrophobic motif (HM). The C-terminal part of this motif also adopts a helical conformation (αHM). (B) Cbk1’s hydrophobic motif (HM) binds residues in interface II of the Cbk1^NTR. The panel shows the view along the axis of αMobA/B and highlights Arg-746 and critical residues at Cbk1^NTR–Mob interface II (Arg-307, Arg-304, and Glu-336). (C) The phosphorylation site on the HM (Thr-743, green) is next to Arg-343 and Arg-344 (yellow) from αMobB, hinting about the importance of Mob coactivator binding in the allosteric phosphoregulation of the Ndr/Lats kinase domain. The HM is shown in stick representation (737-LPFIGYTY-744) or as an α-helical cartoon (745-SRFDYLTKRKNAL-756) in gray. (D) Residues in the interacting region between the HM and the Cbk1^NTR are conserved in human Ndr1/Lats1. Green residues are identical; yellow residues are chemically similar. Numbers in parentheses indicate the human Ndr/Lats residue orthologous to the indicated Cbk1 amino acid (black). (E) Crystallographic model rebuilding also improved the overall quality of the electron density and allowed the Ssd1 docking peptide to be traced (green sticks).

Figure 6.

Cbk1 activation: the role of HM phosphorylation and Mob binding. (A) The activation of AKT/PKB involves a disorder-to-order transition at the αB–αC segment, the activation loop (AL), and the HM. Q220 coordinates the phosphorylated HM (here shown with the S474D phosphomimicking mutation; PDB entry 1O6K). The phosphorylated activation loop (AL; phosphoThr-309) is coordinated by Arg-274 and His-194 (from αC). (B) The inactive Cbk1 structure shows a similar structure apart from the position of its HM. The inactive crystal structure of the Cbk1–Mob2 complex shows that the HM is bound to the groove formed by the Mob-bound NTR and β3 from Cbk1. (C) For activation, the ordering of helix αC in Cbk1 similar to that of AKT/PKB is likely required (MD model). This latter may be supported by interactions formed by phospho-HM and phospho-AL. The activation loop phosphosite (AKT/PKB, Thr-309; Cbk1, Ser-570) is coordinated by an arginine residue from the kinase HRD motif (AKT/PKB, Arg-274; Cbk1, Arg-474) and by another residue from helix αC (AKT/PKB, His-194; Cbk1, His-396). The hydrophobic motif phosphosite (AKT/PKB, Ser-474; Cbk1, Thr-743) in most AGC kinases is coordinated by a single residue from β3 (AKT/PKB, Gln-220). In the case of Cbk1, an arginine residue (Arg-343) from the Mob-bound αMobB coordinates the HM phosphosite while β3 may not be involved. For both AKT/PKB and Cbk1, phospho-HM coordination (by Gln-220-pSer-474 or by Arg-343-pThr-743) brings hydrophobic residues much closer to αC and probably facilitates its ordering. This activation model suggests that coactivator binding is essential not only in HM binding but also in the precise coordination of the kinase’s active state when it is phosphorylated. AKT/PKB is colored yellow, and both HM regions are shown with gray sticks.

Figure 7.

Mechanistic model of activation of the Cbk1–Mob2 complex by the HM and activation loop phosphorylation. The crystal structure of active AKT/PKB (PDB entry 1O6K) highlights the central role of αC in assembling a catalytically competent AGC kinase active site. Glu-200 on αC together with Lys-181 from the N-lobe of the kinase orients the phosphate moiety of ATP (shown in sticks where carbon atoms are colored magenta) and aligns it for substrate phosphorylation involving Asp-275. (The backbone of the substrate peptide bound in the substrate-binding pocket is colored gray.) Activated HM binding in the PIF pocket and activation loop (AL) phosphorylation at Thr-309 buttress αC and promote the proper ATP alignment. The middle panel shows the structural model of the activated Cbk1–Mob2 complex (MD_active_START). The model, which had αC built on the basis of the active AKT/PKB crystallographic model shown in the left panel (but lacked long flexible regions invisible in the crystal structure, e.g., the linker connecting the HM to the kinase domain core or most of the long AL), was created using the revised crystal structure of the nonphosphorylated Cbk1–Mob2 complex. The panel shows the energy-minimized phosphorylated Cbk1–Mob2 structural model and highlights the same key residues in the AGC kinase domain as shown in the AKT/PKB structure on the left. Note that Arg-343 is well-positioned to coordinate phosphoThr-743 on the HM, and this together with phosphoSer570 at the AL affects salt bridge formation between Glu-400 and Lys-381, which in turn could influence active site assembly as in AKT/PKB. The right panel shows the active Cbk1–Mob2 complex after a 25 ns molecular dynamics simulation (MD_active_25 ns).