Merlin, the product of the Nf2 tumor suppressor gene, is an inhibitor of the p21-activated kinase, Pak1.

The Nf2 tumor suppressor gene codes for merlin, a protein whose function has been elusive. We describe a novel interaction between merlin and p21-activated kinase 1 (Pak1), which is dynamic and facilitated upon increased cellular confluence. Merlin inhibits the activation of Pak1, as the loss of merlin expression results in the inappropriate activation of Pak1 under conditions associated with low basal activity.

BRCT repeats as phosphopeptide-binding modules involved in protein targeting.

We used a proteomic approach to identify phosphopeptide-binding modules mediating signal transduction events in the DNA damage response pathway. Using a library of partially degenerate phosphopeptides, we identified tandem BRCT (BRCA1 carboxyl-terminal) domains in PTIP (Pax transactivation domain-interacting protein) and in BRCA1 as phosphoserine- or phosphothreonine-specific binding modules that recognize substrates phosphorylated by the kinases ATM (ataxia telangiectasia-mutated) and ATR (ataxia telangiectasia- and RAD3-related) in response to gamma-irradiation. PTIP tandem BRCT domains are responsible for phosphorylation-dependent protein localization into 53BP1- and phospho-H2AX (gamma-H2AX)-containing nuclear foci, a marker of DNA damage.

The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain.

Polo-like kinases (Plks) perform crucial functions in cell-cycle progression and multiple stages of mitosis. Plks are characterized by a C-terminal noncatalytic region containing two tandem Polo boxes, termed the Polo-box domain (PBD), which has recently been implicated in phosphodependent substrate targeting. We show that the PBDs of human, Xenopus, and yeast Plks all recognize similar phosphoserine/threonine-containing motifs.

Fluorescent caged phosphoserine peptides as probes to investigate phosphorylation-dependent protein associations.

The development of chemical probes for the investigation of the complex phosphorylation signaling cascades that regulate biological events is crucial to understanding these processes. We describe herein a bifunctional probe that enables spatial and temporal release of a biologically active ligand while allowing simultaneous monitoring of its binding to the protein of interest. Substitution of Tyr(-2) for the enviromentally sensitive fluorescent amino acid DANA in the sequence RLYRpSLPA which is known to bind the 14-3-3 protein does not adversely affect binding affinity and allows monitoring of the binding process.

Proteomic identification of 14-3-3zeta as a mitogen-activated protein kinase-activated protein kinase 2 substrate: role in dimer formation and ligand binding.

Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAPK2) mediates multiple p38 MAPK-dependent inflammatory responses. To define the signal transduction pathways activated by MAPKAPK2, we identified potential MAPKAPK2 substrates by using a functional proteomic approach consisting of in vitro phosphorylation of neutrophil lysate by active recombinant MAPKAPK2, protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and phosphoprotein identification by peptide mass fingerprinting with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and protein database analysis. One of the eight candidate MAPKAPK2 substrates identified was the adaptor protein, 14-3-3zeta.

A high-throughput quantitative multiplex kinase assay for monitoring information flow in signaling networks: application to sepsis-apoptosis.

To treat complex human diseases effectively, a systems-level approach is needed to understand the interplay of environmental cues, intracellular signals, and cellular behaviors that underlie disease states. This approach requires high-throughput, multiplex techniques that measure quantitative temporal variations of multiple protein activities in the intracellular signaling network. Here, we describe a single microtiter-based format that simultaneously quantifies protein kinase activities in the phosphatidylinositol 3-kinase pathway (Akt), nuclear factor-kappaB pathway (IKK), and three core mitogen-activated protein kinase pathways (ERK, JNK1, MK2).

Scansite 2.0: Proteome-wide prediction of cell signaling interactions using short sequence motifs.

Scansite identifies short protein sequence motifs that are recognized by modular signaling domains, phosphorylated by protein Ser/Thr- or Tyr-kinases or mediate specific interactions with protein or phospholipid ligands. Each sequence motif is represented as a position-specific scoring matrix (PSSM) based on results from oriented peptide library and phage display experiments. Predicted domain-motif interactions from Scansite can be sequentially combined, allowing segments of biological pathways to be constructed in silico.

Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates.

We have developed a proteomic approach for identifying phosphopeptide binding domains that modulate kinase-dependent signaling pathways. An immobilized library of partially degenerate phosphopeptides biased toward a particular protein kinase phosphorylation motif is used to isolate phospho-binding domains that bind to proteins phosphorylated by that kinase. Applying this approach to cyclin-dependent kinases (Cdks), we identified the polo-box domain (PBD) of the mitotic kinase polo-like kinase 1 (Plk1) as a specific phosphoserine (pSer) or phosphothreonine (pThr) binding domain and determined its optimal binding motif.

Proteomics and systems biology approaches to signal transduction in sepsis.

Sepsis and resulting multiple system organ failure are the leading causes of mortality in intensive care units. Although it is generally appreciated that rampant, deregulated inflammatory pathways play a major role in sepsis, a comprehensive understanding based on the integrated response of multiple signal transduction pathways has remained elusive. Here we review the main signal transduction pathways involved in the progression from inflammation to sepsis and discuss emerging genomic, proteomic, and systems biology approaches to decipher how these signaling pathways converge to cause the septic state.

A novel assay system implicates PtdIns(3,4)P(2), PtdIns(3)P, and PKC delta in intracellular production of reactive oxygen species by the NADPH oxidase.

Activated neutrophils assemble an NADPH oxidase enzyme complex to produce superoxide for microbial killing. Much of the initial oxidase assembly occurs on intracellular granules, followed by movement of the oxidase to phagolysosomes and the plasma membrane. We have developed a novel assay system using Streptolysin-O permeabilized neutrophils that recapitulates the initial intracellular activation process while maintaining the ultrastructural features of this granulocytic cell type.

The p47phox PX domain: two heads are better than one!

The recent X-ray structure of the PX domain of p47phox, a critical subunit of the NADPH oxidase, unexpectedly revealed the presence of two distinct lipid binding pockets within this single modular domain. This unusual feature allows the p47phox PX domain to integrate signal transduction events emerging from two different lipid signaling pathways.

Structural and functional versatility of the FHA domain in DNA-damage signaling by the tumor suppressor kinase Chk2.

The Chk2 Ser/Thr kinase plays crucial, evolutionarily conserved roles in cellular responses to DNA damage. Identification of two pro-oncogenic mutations within the Chk2 FHA domain has highlighted its importance for Chk2 function in checkpoint activation. The X-ray structure of the Chk2 FHA domain in complex with an in vitro selected phosphopeptide motif reveals the determinants of binding specificity and shows that both mutations are remote from the peptide binding site.

Phosphotyrosine-binding domains in signal transduction.

Protein phosphorylation provides molecular control of complex physiological events within cells. In many cases, phosphorylation on specific amino acids directly controls the assembly of multi-protein complexes by recruiting phospho-specific binding modules. Here, the function, structure, and cell biology of phosphotyrosine-binding domains is discussed.

How do 14-3-3 proteins work?-- Gatekeeper phosphorylation and the molecular anvil hypothesis.

14-3-3 proteins were the first signaling molecules to be identified as discrete phosphoserine/threonine binding modules. This family of proteins, which includes seven isotypes in human cells and up to 15 in plants, plays critical roles in cell signaling events that control progress through the cell cycle, transcriptional alterations in response to environmental cues, and programmed cell death. Despite over 30 years of research, distinct roles for most isotypes remain unknown.

Cytoplasmic localization of tristetraprolin involves 14-3-3-dependent and -independent mechanisms.

The immediate early gene tristetraprolin (TTP) is induced transiently in many cell types by numerous extracellular stimuli. TTP encodes a zinc finger protein that can bind and destabilize mRNAs that encode tumor necrosis factor-alpha (TNFalpha) and other cytokines. We hypothesize that TTP also has a broader role in growth factor-responsive pathways.

14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport.

14-3-3 proteins regulate the cell cycle and prevent apoptosis by controlling the nuclear and cytoplasmic distribution of signaling molecules with which they interact. Although the majority of 14-3-3 molecules are present in the cytoplasm, we show here that in the absence of bound ligands 14-3-3 homes to the nucleus. We demonstrate that phosphorylation of one important 14-3-3 binding molecule, the transcription factor FKHRL1, at the 14-3-3 binding site occurs within the nucleus immediately before FKHRL1 relocalization to the cytoplasm.

MAGUK SH3 domains--swapped and stranded by their kinases?

Excitement in protein science comes from unexpected structural findings that shed new light on functional mechanisms. Just such a series of insights is now beginning to emerge from two recently published structures of the scaffolding protein PSD-95.