AKIN10 | SNF1-related protein kinase catalytic subunit alpha KIN10

Arabidopsis AKIN10 but not CPK3 kinase activity is redox?sensitive. (A) In vitro phosphorylation of the 14?3?3 protein?binding site on NIA2 by AKIN10 and CPK3. Kinase reactions were done in the presence of 1 mm DTT or without DTT. Phosphorylation of NIA2 was visualised by western blotting with an antibody recognising a phosphorylated 14?3?3 protein?binding motive only. The coomassie brilliant blue (CBB)?stained membrane is shown below. Positions of the kinases and the substrate are indicated on the right?hand side by black arrowheads and bars. (B) In vitro phosphorylation of bZIP63 by AKIN10. GST?bZIP63 (bZIP63) was incubated with GST?AKIN10 (AKIN10) in kinase buffer containing 32P ?ATP and different concentrations of either DTT or H2O2. The proteins were separated by SDS/PAGE and phosphorylated proteins were detected by subsequent autoradiography. The CBB?stained gel is shown below. The positions of the full?length proteins are indicated. Relative quantification of the signals is shown in Fig. S1B.

AKIN10 T?loop phosphorylation by SnAK2 is redox?dependent. (A) 3D?structural model of the AtSnRK1 complex based on the crystal structure of the rat RnAMPK ?1?1?1 heterotrimeric complex. The left image shows a superposition of the RnAMPK ?1?1?1 crystal structure and the derived AtSnRK1 model. RnAMPK ?1?1?1 is depicted in gold, AtAKIN10 in blue, AtAKIN?1 in red and AtSNF4 in pale?green. The T?loop (yellow) with T198 (green) and C200 (cyan) and the active site (magenta) are highlighted. The image on the right?hand side details the T?loop and active site part of SnRK1 in a sphere?representation with the spheres drawn to represent 1× van der Waals radius (vdwr). (B) In vitro phosphorylation of AKIN10 by SnAK2. Inactive versions of GST?AKIN10 (K48M = AKIN10K/M and T198A = AKIN10T/A) were incubated with its upstream kinase GST?SnAK2 (SnAK2) in kinase buffer containing 32P ?ATP and different concentrations of either DTT or H2O2. The proteins were separated by SDS/PAGE and phosphorylated proteins were detected by subsequent autoradiography. The coomassie brilliant blue (CBB)?stained gel is shown below. The positions of the full?length proteins are indicated by arrowheads. Degradation products of GST?AKIN10 are marked by asterisks. (C) In vitro phosphorylation of the AKIN10 T?loop Threonine (T198) by SnAK2. Wild?type GST?AKIN10 (AKIN10 wt) or the C200S variant (AKIN10 C200S) was incubated with its upstream kinase GST?SnAK2 (SnAK2) in kinase buffer containing different concentrations of either DTT or H2O2. Threonine 198 phosphorylation in the T?loop of AKIN10 was visualised by western blotting with a phospho?specific antibody (??P?AMPK, top). The CBB?stained membrane is shown below.

Intrinsic redox sensitivity of AKIN10 is retained in its T?loop phosphorylated state. (A,B) In vitro kinase activity (A) and phosphorylation (B) of AKIN10 under a simulated H2O2 burst. GST?AKIN10 (AKIN10 or inactive AKIN10K/M = K48M) was first mixed with either its substrate GST?bZIP63 (bZIP63) or its upstream kinase GST?SnAK2 (SnAK2) in kinase buffer containing 3.5 mm GSH. The kinase reactions were then started by adding 32P ?ATP and rising concentrations of H2O2 as indicated. The proteins were separated by SDS/PAGE and phosphorylated proteins were detected via autoradiography. The coomassie brilliant blue (CBB)?stained gel is depicted below. The positions of the full?length proteins are indicated by arrowheads. Degradation products of GST?AKIN10 are marked by asterisks. (C) Redox dependency of in vitro AKIN10 activity before and after phosphorylation by SnAK2. GST?AKIN10 (AKIN10) was first prephosphorylated (left panel) or not (right panel) by SnAK2 in kinase buffer containing 3.5 mm GSH. Then, GST?bZIP63 (bZIP63) and 32P ?ATP were added, either in the continued presence of 3.5 mm GSH (left lane) or in the presence of 3.5 mm GSH + 3.5 mm H2O2 (right lane). bZIP63 phosphorylation was analysed by autoradiography. The CBB?stained gel is depicted below. As activated AKIN10 is ~ 25 times more active than nonactivated AKIN10, the autoradiographs were developed separately in order to avoid oversaturation of the image from activated AKIN10.