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Amy Curran,
Ing-Feng Chang,
Chia-Lun Chang,
Shilpi Garg,
Rodriguez Milla Miguel, Yoshimi D Barron,
Ying Li,
Shawn Romanowsky,
John C Cushman,
Michael Gribskov,
Alice C Harmon,
Jeffrey F Harper
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ABSTRACT: The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca(2+)-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with K(M) ∼70 μM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.
Frontiers in plant science. 01/2011; 2:36.
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ABSTRACT: Calcium signals are critical for the regulation of polarized growth in many eukaryotic cells, including pollen tubes and neurons. In plants, the regulatory pathways that code and decode Ca(2+) signals are poorly understood. In Arabidopsis thaliana, genetic evidence presented here indicates that pollen tube tip growth involves the redundant activity of two Ca(2+)-dependent protein kinases (CPKs), isoforms CPK17 and -34. Both isoforms appear to target to the plasma membrane, as shown by imaging of CPK17-yellow fluorescent protein (YFP) and CPK34-YFP in growing pollen tubes. Segregation analyses from two independent sets of T-DNA insertion mutants indicate that a double disruption of CPK17 and -34 results in an approximately 350-fold reduction in pollen transmission efficiency. The near sterile phenotype of homozygous double mutants could be rescued through pollen expression of a CPK34-YFP fusion. In contrast, a transgene rescue was blocked by mutations engineered to disrupt the Ca(2+)-activation mechanism of CPK34 (CPK34-YFP-E465A,E500A), providing in vivo evidence linking Ca(2+) activation to a biological function of a CPK. While double mutant pollen tubes displayed normal morphology, relative growth rates for the most rapidly growing tubes were reduced by more than three-fold compared with wild type. In addition, while most mutant tubes appeared to grow far enough to reach ovules, the vast majority (>90%) still failed to locate and fertilize ovules. Together, these results provide genetic evidence that CPKs are essential to pollen fitness, and support a mechanistic model in which CPK17 and -34 transduce Ca(2+) signals to increase the rate of pollen tube tip growth and facilitate a response to tropism cues.
The Plant Journal 05/2009; 59(4):528-39. · 6.16 Impact Factor
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Symposia of the Society for Experimental Biology 02/2004;
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ABSTRACT: Protein acetylation is important in regulating DNA-templated processes specifically and protein-protein interactions more generally in eukaryotes. The geminivirus movement protein NSP is essential for virus movement, shuttling the viral DNA genome between the nucleus and the cytoplasm. We have identified a novel Arabidopsis protein, AtNSI, that interacts with NSP. AtNSI is highly conserved among widely divergent plants. Biochemical studies show that its interaction with NSP is direct and that AtNSI acetylates histones, but not NSP, in vitro. Rather, AtNSI specifically acetylates the viral coat protein. AtNSI is a nuclear protein but does not act as a transcriptional coactivator in vitro, which distinguishes it from known eukaryotic histone acetyltransferases. Its overexpression enhances the efficiency of infection by Cabbage leaf curl virus. These findings suggest a role for protein acetylation in coordinating replication of the viral DNA genome with its export from the nucleus.
The Plant Cell 08/2003; 15(7):1605-18. · 8.99 Impact Factor
