Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenviron-ment.

In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endo-somes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half ofthe receptortyrosine kinases in the human genome.

We analyzed a large number phosphorylated proteins in neuroblastoma cells to discern patterns that indicate functional signal transduction pathways.

The analysis revealed that signaling pathways are functionally and physically compartmentalized into distinct collaborative groups distinguished by phosphorylation patterns and intracellular localization.

Src Homology 2 (SH2) domains (and one-fifth of phosphotyrosine-binding or PTB domains) mediate selective protein—protein interactions with proteins phosphorylated on tyrosine residues, and thus mediate assembly of phosphotyrosine signaling networks [19].

Most SH2-SH3 proteins are phosphorylated on multiple sites on tyrosine as well as serine and/or threonine residues.

To identify patterns in tyrosine phosphorylation in neuroblastoma, we acquired phospho-proteomic data from 21 neuroblastoma cell lines and cell fractions including endosomes and detergent-resistant lipid rafts as previously characterized [32,33].

To identify patterns in tyrosine phosphorylation in neuroblastoma, we analyzed tyrosine phos-phoproteomic data acquired from 21 neuroblastoma cell lines using immunoprecipitation of tyrosine phosphorylated peptides as previously described [41,42].

For the first analysis described below, phosphopeptide amounts were summed for each protein in each sample, with the exception of the SRC-family kinases (SFKs), where the C-terminal inhibitory phosphorylation was summed separately and given the names SRC_i; LYN_i; FYN_i; and YESl_i.

This provided an overview of which proteins were present and phosphorylated together in the same samples.

activation occurs in two-steps: RSK_s denotes p9ORSK phosphorylated at a single residue, RSK_d refers to the double phosphorylated , fully active form of p9ORSK (SI and 82 Tables).

The fold change of protein phosphorylation for each treatment condition was calculated in comparison to the respective control and between treatment conditions (Fig 3A).

As eXpected, upon Met inhibitor treatment a strong decrease in phosphorylation of all measured proteins was detected.

By time-resolved quantitative immunoblotting and by protein array we analyzed the phosphorylation of Akt, MEK1/2, ERK1/2 and p9ORSK (SI and S2 Fig).

Akt is activated by phosphorylation on serine 473 and threonine 308 and subsequently phosphorylates multiple substrates with important functions in key biological responses.

Activated Raf leads to phosphorylation of a dual specific kinase, the mitogen-activated protein kinase kinase (MEKI and 2), that phosphorylates the extracellular-signal regulated kinase (ERK1 and 2).

Dual phosphorylated ERK regulates cytoplasmic and nuclear factors and thereby modulates numerous biological responses such as proliferation, differentiation and survival.

A key finding is that, while p53-TAD and its cancer mutants sample a similar set of conformational states, cancer mutants could introduce both local and long-range structural modulations to potentially perturb the balance of p53 binding to various regulatory proteins and further alter how this balance is regulated by multisite phosphorylation of p53-TAD.

The results suggest that, While all sequences sample a similar set of conformational substates, cancer mutants could introduce both local and long-range structural modulations and in turn perturb the balance of p53 binding to various regulatory proteins and further alter how this balance is regulated by multisite phosphorylation of p53-TAD.

Recent NMR and calorimetry studies showed that multisite phosphorylation of TAD reduced binding to MDM2 (by up to 24X, or AAG ~ — 1.9 kcal/mol), and at the same time provided graded enhancement of binding to CBP/p300 domains (by up to 80X, or AAG ~ +2.6 kcal/mol) [66—68].

The graded dependence on the extent of p53 phosphorylation provides a mechanism for gradually increasing p53 response under prolonged genotoxic stress[69].

Nonetheless, precisely how phosphorylation regulates the binding affinities is not entirely clear.

Cellular stresses such as DNA damage, initiate a cascade of phosphorylation events that stabilize and activate the p53 protein [27].