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Polarized growth in pollen tube requires complex signaling events including ROP1 GTPase pathway and calcium signaling. The role of tip calcium in negative feedback regulation of ROP1 remains a question and potentially involves ROP1 negative regulator, REN1GAP, whose activity seems to be regulated by calcium. Calcium-dependent protein kinases (CDPKs/CPKs) are calcium sensors with known function in regulating pollen tube tip growth. As such, we hypothesized that CPK substrate(s) in pollen tube may be component of tip growth regulator such as REN1. Here we report that REN1 is phosphorylated by pollen-expressed CPK16 with a relative EC50 value of ~4.6 µM. MS/MS analysis revealed calcium-dependent phosphorylation sites within REN1 which include Ser70 and Ser267. Functional analyses suggested that REN1 phosphorylation at Ser267 is required for its activity while at Ser70,71 affected its localization and subsequently activity. Mutation analysis of CPK16 loss-of-function, cpk16-3, revealed enhanced pollen tube growth and germination when grown in low calcium media. Treatment of cpk16-3 tubes with either brefeldin A or latrunculin B induced tip swelling phenotype similar to the same effects produced by chemical treatments of partially complemented ren1-1 Lat52::GFP-REN1, Overall, these results suggest a link between calcium and a major signaling pathway, ROP1, via calcium-dependent protein kinase and its substrate REN1. To dissect the causal and phasal relationships between oscillations of growth, active ROP1, F-actin dynamics, and tip-focused calcium, a chemical genetics approach was utilized with the goal to identify small molecules that would specifically activate ROP1. To this end, 20000 chemicals were screened in a cell-based yeast two hybrid assay to target inhibitors of active ROP and GAP. One compound, #7, inhibited ROP-GAP interaction as confirmed by in vitro assays as well as slightly enhanced pollen tube tip width. Treatment of ROP1 OX severely enhanced tip swelli

Polarized growth in pollen tube requires complex signaling events including ROP1 GTPase pathway and calcium signaling. The role of tip calcium in negative feedback regulation of ROP1 remains a question and potentially involves ROP1 negative regulator, REN1GAP, whose activity seems to be regulated by calcium. Calcium-dependent protein kinases (CDPKs/CPKs) are calcium sensors with known function in regulating pollen tube tip growth. As such, we hypothesized that CPK substrate(s) in pollen tube may be component of tip growth regulator such as REN1. Here we report that REN1 is phosphorylated by pollen-expressed CPK16 with a relative EC50 value of ~4.6 µM. MS/MS analysis revealed calcium-dependent phosphorylation sites within REN1 which include Ser70 and Ser267. Functional analyses suggested that REN1 phosphorylation at Ser267 is required for its activity while at Ser70,71 affected its localization and subsequently activity. Mutation analysis of CPK16 loss-of-function, cpk16-3, revealed enhanced pollen tube growth and germination when grown in low calcium media. Treatment of cpk16-3 tubes with either brefeldin A or latrunculin B induced tip swelling phenotype similar to the same effects produced by chemical treatments of partially complemented ren1-1 Lat52::GFP-REN1, Overall, these results suggest a link between calcium and a major signaling pathway, ROP1, via calcium-dependent protein kinase and its substrate REN1. To dissect the causal and phasal relationships between oscillations of growth, active ROP1, F-actin dynamics, and tip-focused calcium, a chemical genetics approach was utilized with the goal to identify small molecules that would specifically activate ROP1. To this end, 20000 chemicals were screened in a cell-based yeast two hybrid assay to target inhibitors of active ROP and GAP. One compound, #7, inhibited ROP-GAP interaction as confirmed by in vitro assays as well as slightly enhanced pollen tube tip width. Treatment of ROP1 OX severely enhanced tip swelli

Calcium-dependent protein kinases (CDPKs) are essential sensor-transducers of calcium signaling pathways in plants. Functional characterization of CDPKs is of great interest because they play important roles during growth, development, and in response to a wide range of environmental stimuli. The Arabidopsis genome encodes 34 CDPKs, but very few substrates of these enzymes have been identified. In this study, we exploited the unique characteristics of CDPKs to develop an efficient approach for the discovery of CDPK-interacting proteins. High-throughput, semi-automated yeast two-hybrid interaction screens with two different cDNA libraries each containing 18 million prey clones were performed using catalytically impaired and constitutively active AtCPK4 and AtCPK11 variants as baits. The use of the constitutively active versions of the CPK baits improved the recovery of positive interacting proteins relative to the wild type kinase. Titration of interaction strength by growth under increasing concentrations of 3-aminotriazole (3-AT), a histidine analog and competitive inhibitor of the His3 gene product, confirmed these results. Possible mechanisms for this observed improvement are discussed. The reproducibility of this approach was assessed by the overlap of several interacting proteins of AtCPK4 and AtCPK11 and the recovery of several putative substrates and indicated that yeast two-hybrid screens using constitutively active and/or catalytically impaired forms of CDPK provides a useful tool to identify potential substrates of the CDPK family and potentially the entire protein kinase superfamily. © 2009 Springer-Verlag.