A cleanup step maximizes the immunoprecipitation of tyrosine-phosphorylated peptides by a conventional antiphosphotyrosine antibody.
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Animals↗
Biomedical subjects
Andrea Gatti
Publications and source records attributed to Andrea Gatti.
Divergence in the upstream signaling of nerve growth factor (NGF) and epidermal growth factor (EGF).
The early response of mammalian cells to growth factors consists of a combination of protein assemblages and kinase cascades. In the study of how signal specificity of different growth factors is determined, PC12 cells represent an elective experimental model to compare the signaling of differentiative nerve growth factor (NGF) and mitogenic epidermal growth factor (EGF). Focusing on Shc as established regulator of the Ras-ERK cascade, whose sustained activation is known to critically shape the neuronal differentiation of PC12 cells, the present manuscript describes two novel sources of divergence in the signaling of NGF and EGF: a differential phosphorylation of p66-Shc at early time points and a differential duration in the recruitment of the overall pool of Shc.
Animals↗
Profiling substrate phosphorylation at the phosphopeptide level.
The identification of substrates is a key aspect in the study of the biological function of protein kinases. The procedure here described is aimed at profiling substrate phosphorylation at the phosphopeptide level by sequentially involving (i). the assessment of the in vitro activity of individual protein kinases on a complex mix of immobilized proteins, (ii). the fractionation of the phosphopeptides being released upon proteolysis of substrates, and (iii). the final identification of the targeted sequences. In particular, the protein sample is spotted onto nitrocellulose membrane and then subjected to a solid-phase kinase assay in the presence of [32P]ATP, prior to solid-phase proteolytic digestion and two-dimensional phosphopeptide mapping. Radiolabeled phosphopeptides are subsequently isolated and sequenced to identify the substrates being targeted by the examined protein kinase. Using the gamma-isotype of p21-activated protein kinase (gamma-PAK) and its known in vitro substrates, I verified that both the specificity of substrate phosphorylation and its efficiency are similar upon solid- and liquid-phase conditions. To demonstrate the feasibility of the overall experimental system, I then employed a fairly crude cell extract as a source of candidate substrates and successfully identified the sequence of a putative substrate of gamma-PAK.
Peptide Mapping↗