Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.phosphorylation ͉ mass spectrometry ͉ strong cation exchange chromatography M uch of eukaryotic protein regulation occurs when protein kinases add a phosphate moiety in an ATP-dependent manner to a Ser, Thr, or Tyr residue of a substrate protein. Not surprisingly, malfunctions in this critical cellular process have been implicated as causal factors in diseases, such as diabetes, cancer, and Alzheimer's. With Ͼ500 identified kinases and thousands of potential substrates, these proteins remain attractive drug targets. Large-scale identification of phosphorylated kinase substrates will certainly enhance our understanding of diverse biological phenomena, potentially leading to targeted intervention in any number of disease paradigms.The identification of phosphorylation sites is most robustly accomplished by MS (1, 2). With tandem MS (MS͞MS), phosphopeptides are fragmented to determine their sequence and to pinpoint the specific Ser, Thr, or Tyr modified by a protein kinase. Despite many reports of thousands of identified proteins from a single biological sample, the large-scale determination of phosphorylation sites is just emerging. To date, the three largest reported repositories of identified sites are from yeast and plant studies [383 (3),125 (4) and Ϸ200 (5)], whereas the most phosphorylation sites identified from a single human sample stands at 64 (6). Clearly, to study the rich biology relying on protein phosphorylation will require more effective methodologies.Uninformative fragmentation is a fundamental obstacle to phosphorylation site analysis, regardless of the scale of an experiment. Fragmentation of phosphopeptides by collision-induced dissociation by MS͞MS commonly results in the production of a single dominant peak corresponding to a neutral loss of phosphoric acid (H 3 PO 4 , 98 Da) from the phosphopeptide (for example, see Fig. 2B). The lack of informative fragmentation at the peptide backbone severely reduces the ability of database searching algorithms to unambiguously identify the phosphopeptide. Furthermore, when a phosphopeptide is identified, it is often ...

An important signaling pathway to the actin cytoskeleton links the Rho family GTPase Cdc42 to the actin-nucleating Arp2/3 complex through N-WASP. Nevertheless, these previously identified components are not sufficient to mediate Cdc42-induced actin polymerization in a physiological context. In this paper, we describe the biochemical purification of Toca-1 (transducer of Cdc42-dependent actin assembly) as an essential component of the Cdc42 pathway. Toca-1 binds both N-WASP and Cdc42 and is a member of the evolutionarily conserved PCH protein family. Toca-1 promotes actin nucleation by activating the N-WASP-WIP/CR16 complex, the predominant form of N-WASP in cells. Thus, the cooperative actions of two distinct Cdc42 effectors, the N-WASP-WIP complex and Toca-1, are required for Cdc42-induced actin assembly. These findings represent a significantly revised view of Cdc42-signaling and shed light on the pathogenesis of Wiskott-Aldrich syndrome.

significant differences between axon-bearing and ax-presented (abstract 291.5) at the 29th Meeting of the 26. A. 8. Ali and A. M. Thomson,]. ~h~s i o l . (london) 507, on-lacking dendrites.Abscisic acid (ABA) stimulates stomatal closure and thus supports water conservation by plants during drought. Mass spectrometry-generated peptide sequence information was used t o clone a Vicia faba complementary DNA, AAPK, encoding a guard cell-specific ABA-activated serine-threonine protein kinase (AAPK). Expression in transformed guard cells of AAPK altered by one amino acid (lysine 43 t o alanine 43) renders stomata insensitive t o ABA-induced closure by eliminating ABA activation of plasma membrane anion channels. This information should allow cell-specific, targeted biotechnological manipulation of crop water status.