Isolation and characterization of kinase interacting protein 1, a pollen protein that interacts with the kinase domain of PRK1, a receptor-like kinase of petunia

Abstract

Many receptor-like kinases have been identified in plants and have been shown by genetic or transgenic knockouts to play diverse physiological roles; however, to date, the cytosolic interacting proteins of relatively few of these kinases have been identified. We have previously identified a predominantly pollen-expressed receptor-like kinase of petunia (Petunia inflata), named PRK1, and we have shown by the antisense RNA approach that it is required for microspores to progress from the unicellular to bicellular stage. To investigate the PRK1-mediated signal transduction pathway, PRK1-K cDNA, encoding most of the cytoplasmic domain of PRK1, was used as bait in yeast (Saccharomyces cerevisiae) two-hybrid screens of pollen/pollen tube cDNA libraries of petunia. A protein named kinase interacting protein 1 (KIP1) was found to interact very strongly with PRK1-K. This interaction was greatly reduced when lysine-462 of PRK1-K, believed to be essential for kinase activity, was replaced with arginine (the resulting protein is named PRK1-K462R). The amino acid sequence of KIP1 deduced from full-length cDNA contains an EF-hand Ca(2+)-binding motif and nine predicted coiled-coil regions. The yeast two-hybrid assay and affinity chromatography showed that KIP1 interacts with itself to form a dimer or higher multimer. KIP1 is present in a single copy in the genome, and is expressed predominantly in pollen with a similar temporal pattern to PRK1. In situ hybridization showed that PRK1 and KIP1 transcripts were localized in the cytoplasm of pollen. PRK1-K phosphorylated KIP1-NT (amino acids 1--716), whereas PRK1-K462R only weakly phosphorylated KIP1-NT in vitro.

Figure 1

Schematic representation of the structural features of full-length and truncated KIP1 (A) and PRK1 (B) proteins. The drawings are to scale. For KIP1, the nine lightly shaded regions denote coiled-coil regions, R denotes the seven tandem repeats of 11 amino acids, and EF (black region) denotes an EF-hand motif (13 amino acids). For PRK1, MS (lightly shaded region) denotes the membrane-spanning domain, T7 denotes the 11-amino acid T7 tag, the five black regions denote the Leu-rich repeats in the extracellular domain, and an asterisk indicates the Lys-462 residue in the cytoplasmic domain that has been replaced with an Arg in PRK1-K462R. The first and last amino acid residues of each peptide are indicated.

Figure 2

RNA gel-blot analysis of expression of KIP1 and PRK1. Twenty micrograms of total RNA was loaded in each lane. The blot was hybridized, stripped of the probe, and reprobed with each of the following cDNAs in succession: KIP1-25, PRK1, and 28S rRNA. Anther-1, Stage 1 anthers from buds that were less than 0.5 cm in length and containing developing microspores in the tetrad configuration; Anther-2, stage 2 anthers from buds between 0.5 and 1.0 cm in length and containing mostly free unicellular microspores; Anther-3, stage 3 anthers from buds between 1.0 and 1.5 cm in length and containing mostly bicellular microspores; Anther-4, stage 4 anthers from buds between 1.5 and 2.0 cm in length and containing essentially all bicellular microspores; Anther-5, stage 5 anthers from purple buds between 2.0 and 2.5 cm in length and containing mature pollen grains. The sizes of KIP1 and PRK1 messages are indicated.

Figure 3

Genomic DNA gel-blot analysis. Each lane contains restriction digests of 12 μg of petunia genomic DNA. Lane 1, EcoRI digest; lane 2, HindIII digest. The blot was probed with KIP1-23 cDNA. Size markers are shown to the right of the blot.

Figure 4

Alignment of the deduced amino acid sequences of KIP1 of petunia and two ORFs of Arabidopsis. AtORF1 corresponds to ORF4 of bacteria artificial chromosome clone F14 m13 (GenBank accession no. AC006592) and encodes 891 amino acids; AtORF2 corresponds to ORF 11 of bacteria artificial chromosome clone F21m12 (GenBank accession no. AC000132) and encodes 947 amino acids. For each aligned position, identical amino acids are shaded in black and conservative changes are shaded in light gray. Hyphens represent gaps that have been introduced to maximize similarity. For KIP1, the nine predicted coiled-coiled regions are overlined; the predicted EF-hand is double-overlined; and the seven 11-amino acid repeats, (A/T)E(G/V)PKNLSTI(K/E), are enclosed in a box. The three boxed regions of AtORF1 correspond to three repeats of 15 amino acids.

Figure 5

Phosphorylation of KIP1 by PRK1-K. GST fusion proteins of PRK1-K, PRK1-K462R, and KIP1-NT were purified by glutathione Sepharose 4B columns and were used for the phosphorylation assay in the presence of [³²P]ATP. Aliquots of five different reaction mixtures, as indicated, were electrophoresed on a 10% (w/v) SDS-polyacrylamide gel that was stained with Coomassie Blue (A) and dried for autoradiography (B). MW, Molecular mass markers.

Figure 6

RNA in situ hybridization of anther sections of petunia. Sections of stage 4 anthers were hybridized with [³⁵S]-labeled KIP1 antisense (A and B), KIP1 sense (C), PRK1 antisense (D and E), and PRK1 sense (F) probes. A, C, D, and F are of the same magnification; bar = 200 μm. B and E are of the same magnification; bar = 50 μm. pg, Pollen grain; aw, anther wall.

Figure 7

Protein gel-blot analysis of the interaction between 6×-His-tag/KIP1-23 and GST/KIP1-23. The blot shown in A was incubated with an anti-GST antibody and the blot shown in B was incubated with a T7-tag monoclonal antibody that reacted with an 11-amino acid sequence present at the N-terminal end of the fusion protein 6×-His-tag/KIP1–23. For both blots, lane 1 represents the fraction eluted from the GST/KIP1-23 affinity column over which total protein extract from E. coli cells harboring the 6×-His-tag/KIP1-23 cDNA construct had been passed, and lane 2 represents the fraction eluted from the GST (+ 22 amino acids from antisense KIP1-23 cDNA) affinity column over which total protein extract from E. coli cells harboring the 6×-His-tag/KIP1–23 cDNA construct had been passed. The protein bands indicated with arrows are GST/KIP1-23 (75 kD) and GST (+ 22 amino acids; 28 kD), and 6×-His-tag/KIP1-23 (48 kD).