Fig. 4.

Activation loop phosphorylation of KrsB is regulated by cAMP. (A) Alignment of human MST1 and D. discoideum KrsB protein sequence surrounding Thr183 in hMST1. (B) Vegetative WT and krsB⁻ cells expressing C-terminally GFP-tagged KrsB constructs or empty vector were lysed and immunoblotted (IB) with an antibody against MST1 phosphorylated on Thr183 (pMST1) or KrsB. The black circle indicates the position of the endogenous KrsB band. (C and D) C-terminally GFP-tagged KrsB constructs expressed in krsB⁻ cells were immunoprecipitated (IP) with antibodies against GFP and subjected to an in vitro kinase assay (KA) in the presence of [γ-³²P]ATP either alone (C) or in various combinations (D). Immunoprecipitates also were immunoblotted with an antibody against GFP. (E) Aggregation-competent WT cells were treated with 1 μM cAMP or vehicle, lysed at the indicated times, and immunoblotted as in B. Densitometric data for phospho-MST1 normalized for the KrsB signal were obtained from four separate experiments and are expressed as mean ± SD. ***P < 0.001.