Source was cell extract of S2 cell line; bait produced from transfected construct; prey produced from transfected construct.

SAK heterodimerizes.

Due to the instability of wild-type SAK, kinase-dead forms of SAK were tested.

Interaction in vitro; enzyme produced as a recombinant fusion protein in bacterial system.

SAK autophosphorylates in vitro.

Interaction in vitro; protein produced as a recombinant fusion protein in bacterial system.

The SAK protein forms a Z-shaped end-to-end dimer.

Interaction in vitro; enzyme produced as a recombinant fusion protein in bacterial system.

SAK autophosphorylates residues within its own slmb degron (residues 292-297) to promote its own degradation. Mutation of the phosphorylation sites stabilizes SAK in S2 cells.

Studies in S2 cells suggest that SAK autophosphorylation can occur in trans. First, kinase-dead GFP-tagged SAK can be phosphorylated at Ser293 and Thr297 by wild-type, Myc tagged SAK. Second, inhibition of SAK dimerization reduces autophosphorylation at Ser293 (but not at Ther297).

Recombinant SAK protein was purified and incubated with ATP. Auto-phosphorylation activity was assayed using phospho-specific antibodies to SAK residues Ser293 and Thr297.

The addition of V5 tagged asl increases SAK dimerization.

Source was cell extract of S2 cell line; bait produced from transfected construct; prey produced from transfected gene.

Interaction in vitro; enzyme produced as a recombinant fusion protein in bacterial system.

SAK autophosphorylates at T172, which is also necessary for subsequent phosphorylation of SAK S293. Additional studies in transfected cells indicates that SAK molecules are autophosphorylated in trans.

Phosphorylation of SAK T172 assessed by phospho-specific antibodies, and confirmed by mass spectrometry.

Two-hybrid system: yeast GAL4-BD/GAL4-AD

Source was yeast cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey was previously cloned reagent).