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The long-awaited structure of HIPK2

  • James M. Murphy
    Correspondence
    To whom correspondence may be addressed: Dr. James M. Murphy, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia. Tel.: 61-3-93452407; E-mail: [email protected]
    Affiliations
    Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia Department of Medical Biology, University of Melbourne, Parkville, Victoria 3052, Australia
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Open AccessPublished:September 13, 2019DOI:https://doi.org/10.1074/jbc.H119.010675
      Homeodomain-interacting protein kinases (HIPKs) are kinases that phosphorylate transcription factors involved in cell proliferation, differentiation, and apoptosis. Their structures have been long sought because of their potential as drug targets in cancers and fibrosis. Agnew and colleagues present the first crystal structure of the HIPK2 kinase domain, complexed with the small-molecule inhibitor CX-4945, revealing important structural differences from related protein kinases of the DYRK family. This structure provides a starting point to exploit HIPK2's distinct structural features to develop selective small-molecule inhibitors of this kinase.

      Introduction

      Protein kinases are essential signal-transducing molecules in all organisms. These enzymes adopt a highly conserved bilobal structure; the N-lobe comprises mostly β-strands and a key regulatory helix (αC), and the larger C-lobe contains mostly helices, with the cleft between lobes where phosphate is transferred from ATP to the protein substrate (
      • Knighton D.R.
      • Zheng J.H.
      • Ten Eyck L.F.
      • Ashford V.A.
      • Xuong N.H.
      • Taylor S.S.
      • Sowadski J.M.
      Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase.
      ). Differences among these kinases in the dispositions and lengths of loops and secondary structure elements are responsible for the differences in how these kinases are regulated and in their abilities to distinguish substrates and partner proteins.
      The HIPK family
      The abbreviations used are: HIPK
      homeodomain-interacting protein kinase
      CK2α
      casein kinase 2 subunit α
      DYRK
      dual-specificity tyrosine-regulated kinase.
      proteins have emerged as promising drug targets in cancers and chronic fibrosis, but their structures have long remained elusive. Agnew et al. (
      • Agnew C.
      • Liu L.
      • Liu S.
      • Xu W.
      • You L.
      • Yeung W.
      • Kannan N.
      • Jablons D.
      • Jura N.
      The crystal structure of the protein kinase HIPK2 reveals a unique architecture of its CMGC-insert region.
      ) now fill this gap by determining the X-ray crystal structure of the kinase domain of HIPK2. This structure provides the first high-resolution glimpse into a HIPK kinase, enabling detailed structural comparisons with the related, and better-understood, dual-specificity tyrosine-regulated protein kinases of the DYRK family.
      It is typical to stabilize active protein kinase conformations to facilitate crystallization by complexing with ATP or analogs. However, here the authors did not obtain crystals when HIPK2 was complexed with ATP or analogs, but only when bound to the casein kinase 2 subunit α (CK2α) inhibitor CX-4945. The structure revealed unique features of the “foot” on the HIPK2 kinase domain, the so-called “CMGC insert,” and a helix unique to the N-lobe of HIPK2 between αC and β-strand 4 (Fig. 1). The CMGC insert is characteristic of CMGC kinases and highly variable among them, serving as a binding region for signaling partner proteins or for oligomerization (
      • Kannan N.
      • Neuwald A.F.
      Evolutionary constraints associated with functional specificity of the CMGC protein kinases MAPK, CDK, GSK, SRPK, DYRK, and CK2α.
      ). This insert arises from an extended sequence connecting αG and αH. Its N terminus contains two short helices (αL and αL′) also present in the closest HIPK2 relatives, the DYRK kinases. In contrast, the adjacent sequence exhibits features unique to HIPK2 (Fig. 1): a β-hairpin longer than that previously observed in DYRK1A and distinct from the loop present in the DYRK2 and DYRK3 structures, a novel helix (termed αM), and a feature more typical of CMGC kinases, termed the αN helix by the authors. Distinct from other CMGC kinases in which this helix is connected to αH via a short loop, the C-terminal end of the CMGC insert contributes to the αH helix, leading to a notably longer helix than that observed in structures of other CMGC family members. As a result, the CMGC insert interacts with the HIPK2 kinase domain C-lobe via a conformation distinctly different from those of other CMGC kinases, including the DYRK family.
      Figure thumbnail gr1
      Figure 1The HIPK2 kinase domain possesses many distinguishing features. The CMGC insert is shown in slate blue, the αC-β4 insert helix in cyan, the N-terminal extension in green, and the small molecule CX-4945 in red. A comprehensive comparison with DYRK kinases is shown in Figs. 4 and 5 of Agnew et al. (
      • Agnew C.
      • Liu L.
      • Liu S.
      • Xu W.
      • You L.
      • Yeung W.
      • Kannan N.
      • Jablons D.
      • Jura N.
      The crystal structure of the protein kinase HIPK2 reveals a unique architecture of its CMGC-insert region.
      ).
      Agnew and colleagues noted two phosphorylation sites in HIPK2: pTyr361 in the activation loop and pSer441 in the CMGC insert (Fig. 1). As in the related DYRK kinases, phosphorylated Tyr361 did not interact with the catalytic loop HRD motif (HAD in HIPK2) and instead engaged a conserved Gln at the base of the substrate-binding pocket. Such an interaction leads to a strained activation loop and is thought to govern substrate switching between Tyr and Ser/Thr. Ser441 is solvent-exposed, positioned in the loop connecting the two strands of the β-hairpin in the CMGC insert. Using molecular dynamics simulations, the authors found that Ser441 phosphorylation does not stabilize the CMGC insert structure. Both pSer441 and the adjacent αM helix, which occludes a protein interaction site (the “P + 3 pocket”) on the C-lobe, likely regulate protein–protein interactions. The authors posit that Ser441 phosphorylation might dictate HIPK2's propensity to dimerize.
      The authors identified additional defining features in the N-lobe of HIPK2. They noted the absence of helical and strand motifs that variously crown the N-lobes of DYRK kinases and also observed a short helix within the loop between αC and β-strand 4 in HIPK2's N-lobe. A sequence analysis revealed that this helix is conserved among the HIPK kinases. The key residue within the αC-β4 insert helix, Tyr258, engaged in interactions with Lys314 in αE and with Glu253 immediately preceding αC. The precise role of the αC-β4 insert helix is currently unclear, but the authors propose that it may regulate the position of the αC helix for catalysis or that it may function as a site for intra- or intermolecular regulatory protein–protein interactions.
      Agnew et al. identified several interesting features within the HIPK2 structure that could not have been predicted by homology with the related DYRK kinases and with CMGC family kinases more broadly. The authors have identified intriguing structural features whose functions remain to be determined, opening up a new area for exploration. Of primary interest, whether these structural elements mediate protein–protein interactions, and thereby contribute to noncatalytic functions of the kinase (
      • Jacobsen A.V.
      • Murphy J.M.
      The secret life of kinases: insights into non-catalytic signalling functions from pseudokinases.
      ,
      • Kung J.E.
      • Jura N.
      Structural basis for the non-catalytic functions of protein kinases.
      ), remains to be established.
      The structure reported by Agnew and colleagues also provides important insights into how HIPK2 might be targeted with small-molecule inhibitors. As noted, HIPK2 was crystallized in complex with the CK2α inhibitor CX-4945, revealing a strong conservation of key compound-binding residues between CK2α and HIPK2. There are some notable differences, however; the Gly-rich loop in HIPK2 is not involved in inhibitor binding, and the CX-4945–binding residue His160 in the active site of CK2α is absent in HIPK2. These differences indicate possible avenues that could be explored by medicinal chemistry to generate selective HIPK2 inhibitors. Such inhibitors would have value in cancer and fibrosis therapies and could provide crucial tools to further explore the physiological functions of HIPK2 in health and disease.

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      Linked Article

      • The crystal structure of the protein kinase HIPK2 reveals a unique architecture of its CMGC-insert region
        Journal of Biological ChemistryVol. 294Issue 37
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          The homeodomain-interacting protein kinase (HIPK) family is comprised of four nuclear protein kinases, HIPK1–4. HIPK proteins phosphorylate a diverse range of transcription factors involved in cell proliferation, differentiation, and apoptosis. HIPK2, thus far the best-characterized member of this largely understudied family of protein kinases, plays a role in the activation of p53 in response to DNA damage. Despite this tumor-suppressor function, HIPK2 is also found overexpressed in several cancers, and its hyperactivation causes chronic fibrosis.
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