IKK2/β (Inhibitor of NF-κB Kinase 2/β) is responsible for phosphorylating IκB (Inhibitor of NF-κB) on Ser-32 and -36, resulting in its proteolytic degradation and activation of the pro-survival transcription factor NF-κB. IKK2/β was originally classified as a serine-specific kinase. However, recently reported in vitro and in vivo studies indicate that tyrosine phosphorylation also plays a role in IKK2/β activity. Upon observing strong tyrosine auto-phosphorylation signals in purified recombinant preparations of human IKK2/β, we previously attempted LC/MS to identify the number and positions of the phosphorylated residues. Other similar studies have identified various phosphorylated residues including Tyr-169, Tyr-188, and Tyr-199. It has also been reported that mutation of Tyr-169 to Phe almost completely abolished the catalytic activity of the enzyme. Tyr-169, which resides within the IKK2/β active site directly after the conserved DFG motif in the activation loop, is unique to IKK1/α and IKK2/β enzymes. Even the close structural homologs TBK1 and IKKκ both contain an Ala residue at this position. Interestingly, neither of these enzymes recognizes both IκB residues Ser-32 and -36 while IKK1/α efficiently carries out this chemistry when incubated with the substrate when assayed in vitro. The amino acid residue corresponding with Tyr-169 in Drosophila melanogaster IKK2 (DmIKK2) is Leu. Despite considerable overlapping functions with its human homolog, the DmIKK2 enzyme fails to phosphorylate residues corresponding to IκB Ser-32 and -36 on the Drosophila IκB homolog Cactus. These observations suggest that the strategic placement of Tyr after the DFG motif in IKK1/α and IKK2/β controls their ability for phosphorylation specificity to IκB on Ser-32 and -36. To test this hypothesis, I have introduced two mutations to convert the activation loop DFGL motif of DmIKK2 to the DLGY sequence present in IKK1/α and IKK2/β. Recombinant baculoviruses were prepared and used to direct recombinant expression of the wild type and mutant DmIKK2 in Sf9 insect cell suspension cultures. The proteins were purified and tested in vitro against human IKK2/β for Tyr auto-phosphorylation and site-specific phosphorylation of IκB.