Fig. 3. Serine to alanine mutations of the predicted cAMP-dependent protein kinase consensus phosphorylation sites in lasp-1 inhibits the phosphorylation of lasp-1 by cAMP-dependent protein kinase in vivo and in vitro. (A) For in vitro analyses, his-tagged wild-type and mutated lasp-1 were phosphorylated and resolved on an SDS-PAGE gel as described in Fig. 2. Inset shows autoradiographic data. Bands were excised from the gel and radiolabel incorporation quantitated by Cerenkov counting (graph). Mutations were as follows: RRDA, Ser99; RGFA, Ser146; R/R, both sites mutated. Values are expressed as a percentage of total counts present in wild-type lasp-1. (B) Western blot analysis of expressed wild-type (WT) and mutated (RGFA¹⁴⁶, RRDA⁹⁹) lasp-1 constructs following transfection of pcDNA3 vectors into MDCK cells. Transfected and mock-transfected (Mock, empty vector) cells were incubated with DMSO vehicle or forskolin (10 µM, 15 minutes) and lysed; extracts were analyzed using the lasp-1 mab as described in Materials and Methods. M_r band shifts are known to correlate with increased phosphorylation in vivo and in vitro (Chew et al., 1998; Chew et al., 2000). (C) Two dimensional western blot analyses of extracts prepared from transfected MDCK cells as described in panel B and Materials and Methods. Note the acidic shift in the RRDA but not the double (R/R) mutant following forskolin stimulation. These data support the conclusion that both Ser99 and Ser146 are in vivo phosphorylation sites.