Kinase experiment:

After serum deprivation for 24 h, cells are incubated with digoxigenin-labeled sham protein or digoxigenin-labeled RV39 at an MOI of 1.0 for 10 min. Cell homogenates are immunoprecipitated with mouse anti-digoxigenin antibody and precipitates incubated with Crosstide and [γ-32P]ATP. Crosstide is a glycogen synthase kinase α/β fusion protein sequence (GRPRTSSFAEG) which is a substrate for Akt. Samples are processed for autoradiography and immunoblotting using rabbit anti-phospho-Tyr416 Src, mouse anti-Src (clone GD11), rabbit anti-phospho-Ser473, or rabbit anti-Akt.

Crosstide is a peptide analog of glycogen synthase kinase α/β fusion protein sequence which is a substrate for Akt.

16HBE14o- cells exposed to RV39 demonstrate a crosstide kinase activity in vitro. Serine phosphorylation of crosstide is confirmed by immunoblotting, and phosphorylation is blocked by PP2 but not by PP3[1]. The wildtype GST-AKT2 shows significant phosphoryl transferase activity towards crosstide, reaching an initial velocity of 16 pmol phosphate/min/μg enzyme. The mutant GST-AKT2T/E,S/D displayed an initial velocity of 85 pmol phosphate/min/μg kinase for phosphorylation of Crosstide, corresponding to a 5-fold increase compared to the wildtype enzyme[2].

[1]. Bentley JK, et al. Rhinovirus activates interleukin-8 expression via a Src/p110beta phosphatidylinositol 3-kinase/Akt pathway in human airway epithelial cells. J Virol. 2007 Feb;81(3):1186-94. Epub 2006 Nov 22. [2]. Baer K, et al. Activation of a GST-tagged AKT2/PKBbeta. Biochim Biophys Acta. 2005 Oct 10;1725(3):340-7. Epub 2005 Apr 20.