Kinase interaction domain of kinase-associated protein phosphatase, a phosphoprotein-binding domain

Abstract

Kinase-associated protein phosphatase interacts specifically with plant receptor-like protein kinases. This interaction is thought to be a key step in signal perception and transduction. The minimal kinase interaction (KI) domain of kinase-associated protein phosphatase was mapped to a 119-aa segment spanning residues 180 to 298. A forkhead-associated (FHA) homology region resides in this minimal KI domain. Site-directed mutagenesis of four highly conserved sites in this FHA homology region abolishes the KI domain's interaction with receptor-like protein kinases, indicating that the FHA region is essential for binding. Serial deletion analysis indicates that 30 aa on each side of the FHA region are also needed for binding; this minimal functional unit is designated as the KI domain. Kinetic studies using surface plasmon resonance indicate that the binding between the KI domain and receptor-like protein kinases has a dissociation constant (KD) of about 25-100 nM, which is similar to the binding affinity of two other well characterized phosphorylation-dependent protein-binding domains (14-3-3 and Src homology 2) and their high-affinity phosphopeptide ligands.

Figure 1

Schematic diagram of KAPP mutants and FHA alignments. (A) KAPP mutants including nine deletion mutants and four site-directed mutants. The largest KAPP fragment, 239 aa in length, was initially identified by interaction cloning (1). The smallest fragment is a 52-aa region with homology to the FHA sequence. (Right) Summary of binding between these KAPP mutants and RLKs from three different binding analyses. (B) Amino acid sequence alignment of the FHA domains from eight representative proteins. The FHA homology region has been identified in many eukaryotic proteins, including transcription factors [FHL1 (26)], kinases [Cds1 (27); DUN1 (28); and RAD53 (29)], phosphatases [KAPPs (1, 2)] and other proteins (fraH, GenBank accession no. U14553). Four highly conserved amino acid residues were chosen for site-directed mutagenesis (∗). Amino acid positions of KAPP were based on the posted sequence from GenBank (accession no. U09505).

Figure 2

Representative binding results from dot-blot immuno analysis. A modified dot-blot immuno analysis was performed to test the binding between GST fusions (KAPP mutants) and MBP fusions (MBP, KIK1 and K558E). Equal amounts (1 μg) of MBP fusion proteins were spotted on the nitrocellulose membrane. The membrane was then cut into many small strips. Each strip, containing three MBP fusion protein spots (MBP, KIK1, and K558E), was incubated with each of the KAPP mutants. The Flag epitope was used to detect the bound GST fusion proteins by chemiluminescent immuno analysis. (A) Coomassie blue-stained SDS/PAGE gel showing the representative KAPP mutant proteins used in the binding experiment. Each lane contained 0.75 μg purified recombinant protein. GST fusion proteins used in these experiments contains a Flag epitope. (B) Immunoblotting results with anti-Flag antibody as the primary antibody and anti-mouse horseradish peroxidase-conjugated IgG as the secondary antibody. Proteins were loaded in the same order as in A. Each lane contains 75 ng of purified protein. (C) Same blot as B. The blot was stripped off and reused for the immunoblot with anti-KAPP antibody as the primary antibody and anti-rabbit peroxydase-labeled IgG as the secondary antibody. (D) Binding results between truncated GST-KAPP fusion proteins and MBP fusions. (E) Binding results between site-directed GST-KAPP mutant proteins and MBP fusions.

Figure 3

Representative binding results between GST fusion proteins (GST, KID119, and KID239) and MBP fusion proteins [MBP (●), RLK5 (○), CLV1 (■), KIK1 (▴), and K558E (▾)] by ELISA. MBP fusion proteins were coated on a 96-well ELISA dish (100 ng/well). Biotinylated GST fusion proteins (40 ng/ml, 200 ng/ml, and 1,000 ng/ml) were incubated with the MBP fusion protein-coated wells overnight. Streptavidin-conjugated alkaline phosphatase and its substrate were used for ELISA color development. (A) Binding results of GST with all five MBP fusion proteins. (B) Binding results of KID119 with all five MBP fusion proteins. (C) Binding of KID 239 with MBP fusion proteins.

Figure 4

Representative SPR sensorgrams of the binding between analytes, GST-KAPP mutants, and ligands, MBP-KIK1 and MBP-CLV1, from BIAcore 2000. The sensorgrams shown here represent the binding between two immobilized MBP fusions (KIK1 and CLV1) at three density levels (illustrated as RU above each curve) and various soluble analytes (all at 25 μg/ml). (A–C Left) Binding results on a KIK1 chip. (D–F) Binding results on a CLV1 chip. Right-most column shows the corresponding analytes for each corresponding row. All the analytes (KAPP mutants) incapable of binding with the two ligands are summarized in A and D. The binding results of KID119 (mapped KI domain) and KID239 are shown in B, C, E, and F, respectively. The corresponding ligand densities in A and D are 258 RU and 1160 RU, respectively. The binding signal from the blank surface of chips has been subtracted from the original results. All curves have been adjusted to the same baseline (zero) to make comparison of sensorgrams easier. Phases before asterisks (∗) represent the association sensorgrams; phases after asterisks represent the dissociation sensorgrams.