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.
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.
Skirpan
A L
AL
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
McCubbin
A G
AG
Ishimizu
T
T
Wang
X
X
Hu
Y
Y
Dowd
P E
PE
Ma
H
H
Kao
T
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018744:DNA, Plant / Q000032:analysis; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011058:Pollen / Q000737:chemistry* / Q000254:growth & development; D017346:Protein Serine-Threonine Kinases; D018749:RNA, Plant / Q000032:analysis; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D015398:Signal Transduction; D019657:Solanaceae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
C436291:CDKN1B protein, human; D002352:Carrier Proteins; D018744:DNA, Plant; D047908:Intracellular Signaling Peptides and Proteins; D010940:Plant Proteins; D018749:RNA, Plant; D050760:Cyclin-Dependent Kinase Inhibitor p27; C088479:PRK1 protein, Petunia inflata; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases