Itro. The recombinant maltosebinding Bryostatin 1 Purity & Documentation protein (MBP) tagged SRK2D (SRK2DMBP) proteins and glutathione Stransferase (GST)tagged CIPK26 (CIPK26GST) proteins showed autophosphorylation activity and protein kinase activity toward myelin standard protein (Supplemental Fig. S4A, lanes 1, 3, 6, and eight). This outcome indicates that both SRK2DMBP and CIPK26GST are functional protein kinases. To clarify irrespective of whether SRK2D and CIPK26 phosphorylate every other, we generated kinaseinactive forms of SRK2D and CIPK26, because their autophosphorylation activities make it difficult to assess the possibility of transphosphorylation events among these protein kinases. Lys42 of CIPK26 and Lys52 of SRK2D correspond to a highly conserved residue that is expected for activity in most protein kinases (Hanks et al., 1988). The Lys42 to Asn mutation of CIPK26 plus the Lys52 to Asn mutation of SRK2D abolished their autophosphorylation activities and their kinase activities toward myelin fundamental protein (Supplemental Fig. S4A, lanes two, four, 7, and 9). This result Chromomycin A3 Cancer confirmed that the mutated GSTtagged CIPK26 (CIPK26K42NGST) proteins along with the mutated MBPtagged SRK2D (SRK2DK52NMBP) proteins had been dysfunctional protein kinases. We carried out in vitro phosphorylation reactions using CIPK26K42NGST or SRK2DK52NMBP as substrate. The SRK2DMBP proteins could not phosphorylate the CIPK26K42NGST proteins (Fig. 3A, lane 3), whereas CIPK26GST proteins phosphorylated SRK2DK52NMBPPlant Physiol. Vol. 167,Protein Kinases in Plant Growth under High Mg2Figure 2. Physical interactions amongst SnRK2s and CIPKs. A, CoIP of CIPK26 with SRK2D in Arabidopsis plants. Transgenic lines harboring both pGH35Spro:43mycCIPK26 and pGK35Spro:SRK2DsGFP or pGK35Spro:sGFP had been subjected to coIP working with antiGFP antibody. Immunoprecipitates had been analyzed by immunoblotting with antiGFP or antimyc antibody. Related results were obtained in independent experiments; representative information are shown. B, Physical interactions amongst CIPK26 and SRK2D derivatives analyzed by yeast twohybrid assay. Representative development status of yeast cells is shown on synthetic dextrose medium agar plates with out Leu, Trp, His, and adenine (SD/2LWHA) with or without the need of 3amino1,two,4triazole (3AT) from duplicate independent trials. Photographs have been taken 7 d right after inoculation. C, Yeast twohybrid assay to validate interaction among CIPK26 and members of SnRK2s. Photographs have been taken 7 d after inoculation. D, BiFC analyses of physical interactions involving SRK2D, SRK2E, or SRK2C and CIPK26 or CIPK24 in N. benthamiana leaves. Transiently transformed N. benthamiana epidermal cells harboring indicated plasmid combinations had been analyzed by confocal microscopy. Yellow fluorescent protein (YFP) fluorescence and Nomarski pictures are shown. Bars = 20 mm. E, Physical interactions among the RD of SRK2D (SRK2D RD) and CIPK26, CIPK3, CIPK9, CIPK23, or CIPK24 analyzed by yeast twohybrid assay. Representative growth status of yeast cells is shown on SD/2LWHA agar medium with 30 mM 3AT from duplicate independent trials. F, BiFC visualization of interaction among SRK2D and CIPK26, CIPK3, CIPK9, and CIPK23 but not CIPK24 in N. benthamiana leaves. The experimental procedure was as described in D. Bars = 50 mm. WB, Western blot.Plant Physiol. Vol. 167, 2015Mogami et al.Figure 3. CIPK26 can phosphorylate SRK2D in vitro. A, Phosphorylation of SRK2DK52NMBP by CIPK26GST in vitro. In vitro phosphorylation assays were carried out with 200 ng of SRK2D (or SRK2DK52N)MBP and 800 ng.
