Figure 4.

T6P Suppresses KIN10 Activation by GRIK1 in Vitro.

(A) In vitro protein phosphorylation assays were performed using purified recombinant KIN10 and GRIK1. Reactions either contained (+) or did not (−) contain 1 µg of GRIK1, 1 µg of KIN10, or 1 mM T6P, Suc, or sorbitol (Sor), as indicated, in the presence of [γ-³²P]ATP. After the reaction, proteins were separated using SDS-PAGE and transferred to PVDF membranes. ³²P-labeled proteins were visualized by autoradiography. Inactive KIN10 kinase mutants, KIN10 (T175A) and KIN10 (M48A), were also included as negative controls. A representative autoradiograph is shown.

(B) KIN10 activity was determined by measuring the incorporation of ³²P from [γ-³²P]ATP into the SnRK1-target peptide of SPS. Kinase activity was quantified for 50 ng of KIN10 or for a mixture of 50 ng of KIN10 + 50 ng of GRIK1 or for 50 ng of GRIK1, in the absence (−) or presence of 1 mM T6P or Suc. Values represent mean ± sd of three independent replicates. Levels indicated with different letters above histogram bars are significantly different (Student’s t test for all pairs of the same proteins, P < 0.05; Supplemental File 1). DPM, disintegrations per minute.