ReviewBiochemistry

Revisiting protein kinase–substrate interactions: Toward therapeutic development

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Science Signaling  22 Mar 2016:
Vol. 9, Issue 420, pp. re3
DOI: 10.1126/scisignal.aad4016

Figures

  • Fig. 1

    Structure of the catalytic domain of protein kinases. (A) Classical division of the catalytic domain, showing a small N-terminal lobe (blue) and the C-terminal lobe (red). (B) Same structure as in (A) highlighting the functional aspects of the catalytic domain. The ATP binding site (green) is present in the cleft between the lobes. The SBS (yellow) interacts directly with the substrate (purple), aiding the selectivity process. PKI, protein kinase inhibitor.

  • Fig. 2

    Examples of protein kinases interacting with substrates with linear and structurally formed consensus sites. (A) Crystal structure of PKA (PDB: 3FJQ; gray) (16) interacting with a pseudosubstrate peptide that presents the linear consensus (yellow). The red spots on the surface of PKA highlight acidic residues that interact with the P-2 and P-3 positions of the substrate (blue sticks, both arginines), which determine the PKA/PKC consensus phosphorylation motif as [K/R][K/R]X[S/T]. The purple spot on the surface denotes the catalytic residue, and the orange mark on the substrate denotes the position of the phosphorylated residue (P0). (B) Model of PKC (gray) interacting with a known substrate (α-tubulin, blue). Acidic residues that interact with the basic residues on the substrate are depicted in the SBS (red). The phosphorylatable residue, Thr253 (orange) of α-tubulin is not found in a PKA/PKC phosphorylation motif. Structural analyses of this substrate revealed that basic residues Lys163 and Lys164 (blue sticks) are close to Thr253, presenting a spatial conformation similar to the linear substrate.

  • Fig. 3

    Examples of protein kinases interacting through the docking site. (A) Structure of protein kinase p38α (gray; PDB: 3GC7) (122) with the docking site highlighted (orange). Three peptides of substrates from different MAPKs are overlapped to show small differences in physical interactions that determine specificity: in blue, a peptide from p38α substrate MK2 (PDB: 2OKR) (123); in red, a peptide from ERK1 substrate, MNK1 (PDB: 2Y9Q) (124); and in green, a peptide from JNK1 substrate, NFAT4 (PDB: 2XRW) (124). The peptide in pink is a spatial reference for the location of the SBS, showing the distance between these two key interaction sites in p38α. (B) Structure of p38α interacting with phosphatase MKP5 (pink) compared to the interaction with the linear peptide from a p38α substrate (blue), showing that the interaction on the docking site also presents the possibility of “structural consensus specificity,” which can be relevant for substrate specificity.

  • Fig. 4

    Different regions (mainly N-lobe) of the catalytic domain of protein kinases explored as drug binding sites. (A) The ATP pocket is targeted by type 1 inhibitors (PDB: 1FMO) (88). (B) A pocket formed in the DFG-out conformation is targeted by type 2 inhibitors, such as imatinib [PDB: 2HYY (125)], depicted in darker blue sticks. (C) Type 3 inhibitors target a hydrophobic pocket (but not the ATP binding region) released in DFG-out conformations. For example, depicted in darker red sticks within the red hydrophobic pocket is the non–ATP-competitive inhibitor N-{4-[(1S)-1,2-dihydroxyethyl]benzyl}-N-methyl-4-(phenylsulfamoyl)benzamide of human LIMK2 kinase domain (PDB: 4TPT) (126). (D) A pocket formed in the surface of the N-lobe of MEK1 binds the non–ATP-competitive inhibitor 2-([3R-3,4-dihydroxybutyl]oxy)-4-fluoro-6-[(2-fluoro-4-iodophenyl)amino]benzamide (PDB: 4ARK) (127). (E) A shallow crevice and ATP binding pocket are occupied by an inhibitor formed by a synthetic peptide linked to thiophosphoric acid o-((adenosyl-phospho)phospho)-s-acetamidyl-diester, a typical type 4 inhibitor (magenta sticks; PDB: 1GAG) (117). (F) General view of all surfaces of pockets used by different inhibitor types. The reference structure (gray) is PDB: 2HYY (125).

Tables

  • Table 1. Biochemical methods used to detect protein kinase substrates.

    Listed are various methods using biochemical approaches to predict substrates on the basis of linear and structural motifs.

    Biochemical
    methods
    Arrangement of detected motif in detected
    substrate
    References
    Two-dimensional gel electrophoresis and mass spectrometryLinear and structural motifs(40)
    Stable isotope labeling by amino acids in cell culture (SILAC)Linear and structural motifs(41, 42, 53, 128)
    Immunoprecipitation and mass spectrometry (kinase-specific antibodies)Linear and structural motifs(129)
    Immunoprecipitation and mass spectrometry (antibodies against phosphomotifs)Linear and structural motifs(54)
    Yeast two-hybrid (Y2H) systemLinear and structural motifs(61, 62)
    Split-ubiquitin system (SUS)Linear and structural motifs(64, 65)
    Bimolecular fluorescence complementation (BiFC)Linear and structural motifs(65, 66)
    Kinase-interacting substrate screening (KISS)Linear and structural motifs(60)
    Kinase assay linked with phosphoproteomics (KALIP)Linear motifs(67)
    Engineered kinases (chemical genetics)Linear and structural motifs(70, 71)
  • Table 2. Computational methods used to predict protein kinase substrates.

    Listed are various methods using computational tools to predict substrates on the basis of sequence or structure.

    Computational methodsBasis of the search spaceSearch windowReference
    NetPhosSequence9–33 residues(74)
    PredkinSequence7 residues(76)
    NetphosKSequence9–33 residues(75)
    DISPHOSSequence25 residues(80)
    pkaPSSequence80 residues(82)
    NetworKINSequence9–33 residues(83)
    Unnamed methodSequence9 residues(81)
    Predkin 2.0Sequence7 residues(77)
    Phos3DStructure2 to 10 Å(85)
    Unnamed methodStructure3 to 12 Å(86)

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