Fig. 2. Time course of phosphorylation of lasp-1 by cAMP-dependent protein kinase and tentative identification of the major in vitro phosphorylation sites. (A) Top panel: diagram showing location of the two known cAMP-dependent protein kinase consensus sites in nebulin repeat region. Lower panel: time course of phosphorylation of his-tagged lasp-1 by a recombinant subunit of cAMP-dependent protein kinase (see Materials and Methods for details). (B) Chromatogram of tryptic digest of lasp-1. 20 µg of his-tagged lasp-1 was phosphorylated with recombinant catalytic subunit of cAMP-dependent protein kinase using [-³²P]ATP as a substrate. Radiolabeled protein was isolated on an SDS-PAGE gel, subjected to in-gel tryptic digest and labeled peptides resolved on a SMART system with an acetonitrile gradient (flow rate, 100 µl/minute). Peaks shown in the figure were further resolved using a slower flow rate (50 µl/minute) then microsequenced. Parentheses in the sequences locate the predicted position of arginine and other residues present in the deduced lasp-1 sequence. Since trypsin normally cleaves at arginine residues, these amino acids were not expected, nor were they identified in sequencing analyses. However, because it is known that trypsin does not readily cleave R-X-Ser(P), it is likely that the tryptic fragment in the second peak contained the RRDS sequence in lower abundance compared with the MGPSGGEGAEPE fragment.