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Alterations in cellular phosphorylation patterns have been implicated in a number of diseases, including cancer, through multiple mechanisms. Herein we present a survey of the phosphorylation profiles of an isogenic pair of human cancer cell lines with opposite metastatic phenotype. Phosphopeptides were enriched from tumor cell lysates with titanium dioxide and zirconium dioxide, and identified with nano-LC-MS/MS using an automatic cross-validation of MS/MS and MS/MS/MS (MS2+MS3) data-dependent neutral loss method. A spectral counting quantitative strategy was applied to the two cell line samples on the MS2-only scan, which was implemented successively after each MS2+MS3 scan in the same sample. For all regulated phosphopeptides reported by spectral counting analysis, sequence and phosphorylation site assignments were validated by a MS2+MS3 data-dependent neutral loss method. With this approach, we identified over 70 phosphorylated sites on 27 phosphoproteins as being differentially expressed with respect to tumor cell phenotype. The altered expression levels of proteins identified by LC-MS/MS were validated using Western blotting. Using network pathway analysis, we observed that the majority of the differentially expressed proteins were highly interconnected and belong to two major intracellular signaling pathways. Our findings suggest that the phosphorylation of isoform A of lamin A/C and GTPase activating protein binding protein 1 is associated with metastatic propensity. The study demonstrates a quantitative and comparative proteomics strategy to identify differential phosphorylation patterns in complex biological samples.

Microglial cells in the brains of Alzheimer's patients are known to be recruited to amyloid-beta (Aβ) plaques where they exhibit an activated phenotype, but are defective for plaque removal by phagocytosis. In this study, we show that microglia stressed by exposure to sodium arsenite or Aβ(1–42) peptides or fibrils form extensive stress granules (SGs) to which the tyrosine kinase, SYK, is recruited. SYK enhances the formation of SGs, is active within the resulting SGs and stimulates the production of reactive oxygen and nitrogen species that are toxic to neuronal cells. This sequestration of SYK inhibits the ability of microglial cells to phagocytose Escherichia coli or Aβ fibrils. We find that aged microglial cells are more susceptible to the formation of SGs; and SGs containing SYK and phosphotyrosine are prevalent in the brains of patients with severe Alzheimer's disease. Phagocytic activity can be restored to stressed microglial cells by treatment with IgG, suggesting a mechanism to explain the therapeutic efficacy of intravenous IgG. These studies describe a mechanism by which stress, including exposure to Aβ, compromises the function of microglial cells in Alzheimer's disease and suggest approaches to restore activity to dysfunctional microglial cells., Graphical Abstract, Highlights • Chronic stress promotes the formation of large, persistent stress granules in microglial cells. • SYK is recruited to stress granules, which promotes inflammatory responses and inhibits phagocytosis. • Phagocytic activity of stressed cells can be recovered by treatment with IgG. Microglial cells in the brains of patients with Alzheimer's disease are activated, but are defective at phagocytosis of amyloid plaques. Activation and phagocytosis require the SYK tyrosine kinase. Chronic exposure to amyloid-beta promotes the formation of persistent stress granules to which active SYK binds and these are found in the brains of patients with severe Alzheimer's disease. This activation and sequestration of SYK promotes inflammation and inhibits phagocytosis. Phagocytic activity can be recovered by treatment with IgG, which causes a redistribution of SYK within the cell, suggesting potential therapeutic approaches to restoring microglial cell function to diseased or aged brains.

We have investigated the proteinase-inhibitory activity against cathepsin L of some c-Ha-ras gene products (p21s) with point mutations at position 12, 33 or 35, which affect the interaction with GTPase-activating protein (GAP). All of the full-length p21s examined showed similar inhibitory activities irrespective of the point mutation and the transforming activity. These results suggest that mutations at GAP-interacting sites have no effect on the proteinase-inhibitory activity of p21s and that the proteinase-inhibitory activity alone is not sufficient for the transformation caused by p21s.