Phosphorylation analysis of GRF4 by MPK3 and MPK6

The coding sequences of MKK5^T215D/S221D (MKK5^DD) (48 (link)), MPK3, MPK6, GRF4, and GRF4^S248A were cloned into the pGEX-4T-1 vector to add a GST tag. The plasmids were transformed into E. coli BL21, and the production of recombinant GST-MKK5^DD, GST-MPK3, GST-MPK6, GST-GRF4, and GST-GRF4^S248A was induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside at 16°C for 12 hours and then purified with Glutathione Sepharose 4 Fast Flow (GE Healthcare, Chicago, IL, USA). The in vitro kinase assay was performed as described previously (49 (link)). In brief, 0.1 μg of recombinant GST-MKK5^DD, 0.2 μg of GST-MPK3 or GST-MPK6, and 2 μg of GST-GRF4 or GST-GRF4^S248A were incubated in a 30-μl kinase reaction [10 mM MgCl₂, 1 mM adenosine 5′-O-(3-thiotriphosphate), 50 mM tris-HCl (pH 7.5), and 1 mM DTT] at 23°C for 30 min. Then, 20 mM EDTA and 1.5 mM p-nitrobenzyl mesylate (ABclonal, Wuhan, China) were added and incubated at 23°C for another 30 min, and 5× loading buffer was used to terminate the reaction. Anti-TPE (ABclonal, Wuhan, China), an antibody that specifically recognizes phosphorylated proteins (53 (link), 54 (link)), was used to determine whether MPK3 and MPK6 can phosphorylate GRF4 and GRF4^S248A, and the anti-GST antibody (TransGen, Beijing, China) was used to determine whether the loading quantity of each protein was consistent.