Correction: Intestinal epithelial cell-specific RARα depletion results in aberrant epithelial cell homeostasis and underdeveloped immune system.

The original version of this Article omitted the author Margarita Parada-kusz from the Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA, USA.

Blood clotting and traumatic injury with shock mediates complement-dependent neutrophil priming for extracellular ROS, ROS-dependent organ injury and coagulopathy.

Polymorphonuclear (PMN) leucocytes participate in acute inflammatory pathologies such as acute respiratory distress syndrome (ARDS) following traumatic injury and shock, which also activates the coagulation system systemically. Trauma can prime the PMN nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex for an enhanced respiratory burst, but the relative role of various priming agents in this process remains incompletely understood. We therefore set out to identify mediators of PMN priming during coagulation and trauma-shock and determine whether PMN reactive oxygen species (ROS) generated in this manner could influence organ injury and coagulation. Initial experiments demonstrated that PMN are primed for predominantly extracellular ROS production by products of coagulation, which was abrogated by CD88/C5a receptor(C5aR) inhibition. The importance of this was highlighted further by demonstrating that known PMN priming agents result in fractionally different amounts of extracellular versus intracellular ROS release depending on the agent used. Plasma from trauma patients in haemodynamic shock (n = 10) also primed PMN for extracellular ROS in a C5a-dependent manner, which correlated with both complement alternative pathway activation and thrombin generation. Furthermore, PMN primed by preincubation with products of blood coagulation directly caused loss of endothelial barrier function in vitro that was abrogated by C5aR blockade or NADPH oxidase inhibition. Finally, we show in a murine model of trauma-shock that p47phox knock-out (KO) mice with PMN incapable of generating ROS were protected from inflammatory end-organ injury and activated protein C-mediated coagulopathy. In summary, we demonstrate that trauma-shock and coagulation primes PMN for predominantly extracellular ROS production in a C5a-dependent manner that contributes to endothelial barrier loss and organ injury, and potentially enhances traumatic coagulopathy.

Intestinal epithelial cell-specific RARα depletion results in aberrant epithelial cell homeostasis and underdeveloped immune system.

Retinoic acid (RA), a dietary vitamin A metabolite, is crucial in maintaining intestinal homeostasis. RA acts on intestinal leukocytes to modulate their lineage commitment and function. Although the role of RA has been characterized in immune cells, whether intestinal epithelial cells (IECs) rely on RA signaling to exert their immune-regulatory function has not been examined. Here we demonstrate that lack of RA receptor α (RARα) signaling in IECs results in deregulated epithelial lineage specification, leading to increased numbers of goblet cells and Paneth cells. Mechanistically, lack of RARα resulted in increased KLF4+ goblet cell precursors in the distal bowel, whereas RA treatment inhibited klf4 expression and goblet cell differentiation in zebrafish. These changes in secretory cells are associated with increased Reg3g, reduced luminal bacterial detection, and an underdeveloped intestinal immune system, as evidenced by an almost complete absence of lymphoid follicles and gut resident mononuclear phagocytes. This underdeveloped intestinal immune system shows a decreased ability to clear infection with Citrobacter rodentium. Collectively, our findings indicate that epithelial cell-intrinsic RARα signaling is critical to the global development of the intestinal immune system.

Canonical Wnt signalling activates TAZ through PP1A during osteogenic differentiation.

TAZ, a transcriptional modulator, has a key role in cell proliferation, differentiation and stem cell self-renewal. TAZ activity is regulated by several signalling pathways, including Hippo, GPCR and Wnt signalling, but the regulatory mechanisms of TAZ activation are not yet clearly understood. In this report, we show that TAZ is regulated by canonical Wnt signalling during osteogenic differentiation. Wnt3a increases TAZ expression and an inhibitor of GSK3ß, a downstream effector of Wnt signalling, induces TAZ. Wnt3a facilitates the dephosphorylation of TAZ, which stabilises TAZ and prevents it from binding 14-3-3 proteins, thus inducing the nuclear localisation of TAZ. Dephosphorylation of TAZ occurs via PP1A, and depletion of PP1A blocks Wnt3a-induced TAZ stabilisation. Wnt3a-induced TAZ activates osteoblastic differentiation and siRNA-induced TAZ depletion decreases Wnt3a-induced osteoblast differentiation. Taken together, these results show that TAZ mediates Wnt3a-stimulated osteogenic differentiation through PP1A, suggesting that the Wnt signal regulates the Hippo pathway.

Structural basis for the interaction of the free SH2 domain EAT-2 with SLAM receptors in hematopoietic cells.

The T and natural killer (NK) cell-specific gene SAP (SH2D1A) encodes a 'free SH2 domain' that binds a specific tyrosine motif in the cytoplasmic tail of SLAM (CD150) and related cell surface proteins. Mutations in SH2D1A cause the X-linked lymphoproliferative disease, a primary immunodeficiency. Here we report that a second gene encoding a free SH2 domain, EAT-2, is expressed in macrophages and B lympho cytes. The EAT-2 structure in complex with a phosphotyrosine peptide containing a sequence motif with Tyr281 of the cytoplasmic tail of CD150 is very similar to the structure of SH2D1A complexed with the same peptide. This explains the high affinity of EAT-2 for the pTyr motif in the cytoplasmic tail of CD150 but, unlike SH2D1A, EAT-2 does not bind to non-phosphorylated CD150. EAT-2 binds to the phosphorylated receptors CD84, CD150, CD229 and CD244, and acts as a natural inhibitor, which interferes with the recruitment of the tyrosine phosphatase SHP-2. We conclude that EAT-2 plays a role in controlling signal transduction through at least four receptors expressed on the surface of professional antigen-presenting cells.

Phosphorylation and cell cycle-dependent regulation of Na+/H+ exchanger regulatory factor-1 by Cdc2 kinase.

Na(+)/H(+) exchanger regulatory factor (NHERF)-1 is a PDZ domain-containing adaptor protein known to bind to various receptors, channels, cytoskeletal elements, and cytoplasmic signaling proteins. We report here that the phosphorylation state of NHERF-1 is profoundly regulated by the cell cycle: NHERF-1 in HeLa cells is hyperphosphorylated in mitosis phase and much less phosphorylated at other points of the cell cycle. This mitosis phase-dependent phosphorylation of NHERF-1 could be blocked by roscovitine, consistent with phosphorylation by cyclin-dependent kinases. In vitro studies with purified NHERF-1 fusion proteins and purified kinases revealed that NHERF-1 was robustly phosphorylated by the cyclin-dependent kinase Cdc2. In contrast, the NHERF-1 relative NHERF-2 was not phosphorylated at all by Cdc2. NHERF-1 possesses two serines (Ser(279) and Ser(301)) that conform to the SPX(K/R) motif preferred for phosphorylation by Cdc2. Mutation of either of these serines reduced Cdc2-mediated phosphorylation of NHERF-1 in vitro, and mutation of both residues together completely abolished Cdc2-mediated phosphorylation. When the S279A/S301A NHERF-1 mutant was expressed in cells, it failed to exhibit the mitosis phase-dependent phosphorylation observed with wild-type NHERF-1. Mutation of both Ser(279) and Ser(301) to aspartate, to mimic Cdc2 phosphorylation of NHERF-1, resulted in a NHERF-1 mutant with a markedly impaired ability to oligomerize in vitro. Similarly, endogenous NHERF-1 from lysates of mitosis phase HeLa cells exhibited a markedly reduced ability to oligomerize relative to endogenous NHERF-1 from lysates of interphase HeLa cells. Mitosis phase NHERF-1 furthermore exhibited the ability to associate with Pin1, a WW domain-containing peptidylprolyl isomerase that does not detectably bind to NHERF-1 in interphase lysates. The association of NHERF-1 with Pin1 facilitated dephosphorylation of NHERF-1, as shown in experiments in which cellular Pin1 activity was blocked by the selective inhibitor juglone. These data reveal that cellular NHERF-1 is phosphorylated during mitosis phase by Cdc2 at Ser(279) and Ser(301) and that this phosphorylation regulates NHERF-1 oligomerization and association with Pin1.

The PX domains of p47phox and p40phox bind to lipid products of PI(3)K.

PX domains are found in a variety of proteins that associate with cell membranes, but their molecular function has remained obscure. We show here that the PX domains in p47phox and p40phox subunits of the phagocyte NADPH oxidase bind to phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P(2)) and phosphatidylinositol-3-phosphate (PtdIns(3)P), respectively. We also show that an Arg-to-Gln mutation in the PX domain of p47phox, which is found in patients with chronic granulomatous disease, eliminates phosphoinositide binding, as does the analogous mutation in the PX domain of p40phox. The PX domain of p40phox localizes specifically to PtdIns(3)P-enriched early endosomes, and this localization is disrupted by inhibition of phosphoinositide-3-OH kinase (PI(3)K) or by the Arg-to-Gln point mutation. These findings provide a molecular foundation to understand the role of PI(3)K in regulating neutrophil function and inflammation, and to identify PX domains as specific phosphoinositide-binding modules involved in signal transduction events in eukaryotic cells.

A motif-based profile scanning approach for genome-wide prediction of signaling pathways.

The rapid increase in genomic information requires new techniques to infer protein function and predict protein-protein interactions. Bioinformatics identifies modular signaling domains within protein sequences with a high degree of accuracy. In contrast, little success has been achieved in predicting short linear sequence motifs within proteins targeted by these domains to form complex signaling networks. Here we describe a peptide library-based searching algorithm, accessible over the World Wide Web, that identifies sequence motifs likely to bind to specific protein domains such as 14-3-3, SH2, and SH3 domains, or likely to be phosphorylated by specific protein kinases such as Src and AKT. Predictions from database searches for proteins containing motifs matching two different domains in a common signaling pathway provides a much higher success rate. This technology facilitates prediction of cell signaling networks within proteomes, and could aid in the identification of drug targets for the treatment of human diseases.

PhosphoSerine/threonine binding domains: you can't pSERious?

The fundamental biological importance of protein phosphorylation is underlined by the existence of more than 500 protein kinase genes within the human genome. In many cases, phosphorylation on serine, threonine, and tyrosine residues creates binding surfaces for a variety of phospho-amino acid binding proteins/modules. Here, we review the insights into serine/threonine phosphorylation-dependent signal transduction processes provided by structures of several of these proteins and their complexes.

Phosphoserine/threonine-binding domains.

Phosphorylation of proteins on serine and threonine residues has traditionally been viewed as a means to allosterically regulate catalytic activity. Research within the past five years, however, has revealed that serine/threonine phosphorylation can also directly result in the formation of multimolecular signaling complexes through specific interactions between phosphoserine/threonine (pSer/Thr)-binding modules and phosphorylated sequence motifs. pSer/Thr-binding proteins and domains currently include 14-3-3, WW domains, forkhead-associated domains, and, tentatively, WD40 repeats and leucine-rich regions. It seems likely that additional modules will be found in the future. The amino acid sequences recognized by these pSer/Thr-binding modules show partial overlap with the optimal phosphorylation motifs for different protein kinase subfamilies, allowing the formation of specific signaling complexes to be controlled through combinatorial interactions between particular upstream kinases and a particular binding module. The structural basis for pSer/Thr binding differs dramatically between 14-3-3 proteins, WW domains and forkhead-associated domains, suggesting that their pSer/Thr binding function was acquired through convergent evolution.

The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms.

Forkhead-associated (FHA) domains are a class of ubiquitous signaling modules that appear to function through interactions with phosphorylated target molecules. We have used oriented peptide library screening to determine the optimal phosphopeptide binding motifs recognized by several FHA domains, including those within a number of DNA damage checkpoint kinases, and determined the X-ray structure of Rad53p-FHA1, in complex with a phospho-threonine peptide, at 1.6 A resolution. The structure reveals a striking similarity to the MH2 domains of Smad tumor suppressor proteins and reveals a mode of peptide binding that differs from SH2, 14-3-3, or PTB domain complexes. These results have important implications for DNA damage signaling and CHK2-dependent tumor suppression, and they indicate that FHA domains play important and unsuspected roles in S/T kinase signaling mechanisms in prokaryotes and eukaryotes.

TAZ: a novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins.

The highly conserved and ubiquitously expressed 14-3-3 proteins regulate differentiation, cell cycle progression and apoptosis by binding intracellular phosphoproteins involved in signal transduction. By screening in vitro translated cDNA pools for the ability to bind 14-3-3, we identified a novel transcriptional co-activator, TAZ (transcriptional co-activator with PDZ-binding motif) as a 14-3-3-binding molecule. TAZ shares homology with Yes-associated protein (YAP), contains a WW domain and functions as a transcriptional co-activator by binding to the PPXY motif present on transcription factors. 14-3-3 binding requires TAZ phosphorylation on a single serine residue, resulting in the inhibition of TAZ transcriptional co-activation through 14-3-3-mediated nuclear export. The C-terminus of TAZ contains a highly conserved PDZ-binding motif that localizes TAZ into discrete nuclear foci and is essential for TAZ-stimulated gene transcription. TAZ uses this same motif to bind the PDZ domain-containing protein NHERF-2, a molecule that tethers plasma membrane ion channels and receptors to cytoskeletal actin. TAZ may link events at the plasma membrane and cytoskeleton to nuclear transcription in a manner that can be regulated by 14-3-3.

A peptide library approach identifies a specific inhibitor for the ZAP-70 protein tyrosine kinase.

We utilized a novel peptide library approach to identify specific inhibitors of ZAP-70, a protein Tyr kinase involved in T cell activation. By screening more than 6 billion peptides oriented by a common Tyr residue for their ability to bind to ZAP-70, we determined a consensus optimal peptide. A Phe-for-Tyr substituted version of the peptide inhibited ZAP-70 protein Tyr kinase activity by competing with protein substrates (K(I) of 2 microM). The related protein Tyr kinases, Lck and Syk, were not significantly inhibited by the peptide. When introduced into intact T cells, the peptide blocked signaling downstream of ZAP-70, including ZAP-70-dependent gene induction, without affecting upstream Tyr phosphorylation. Thus, screening Tyr-oriented peptide libraries can identify selective peptide inhibitors of protein Tyr kinases.

14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation.

The Bcl-2 homology 3 (BH3) domain of prodeath Bcl-2 family members mediates their interaction with prosurvival Bcl-2 family members and promotes apoptosis. We report that survival factors trigger the phosphorylation of the proapoptotic Bcl-2 family member BAD at a site (Ser-155) within the BAD BH3 domain. When BAD is bound to prosurvival Bcl-2 family members, BAD Ser-155 phosphorylation requires the prior phosphorylation of Ser-136, which recruits 14-3-3 proteins that then function to increase the accessibility of Ser-155 to survival-promoting kinases. Ser-155 phosphorylation disrupts the binding of BAD to prosurvival Bcl-2 proteins and thereby promotes cell survival. These findings define a mechanism by which survival signals inactivate a proapoptotic Bcl-2 family member, and suggest a role for 14-3-3 proteins as cofactors that regulate sequential protein phosphorylation events.

Peptide and protein library screening defines optimal substrate motifs for AKT/PKB.

AKT was originally identified as a proto-oncogene with a pleckstrin homology and Ser/Thr protein kinase domains. Recent studies revealed that AKT regulates a variety of cellular functions including cell survival, cell growth, cell differentiation, cell cycle progression, transcription, translation, and cellular metabolism. To clarify the substrate specificity of AKT, we have used an oriented peptide library approach to determine optimal amino acids at positions N-terminal and C-terminal to the site of phosphorylation. The predicted optimal peptide substrate (Arg-Lys-Arg-Xaa-Arg-Thr-Tyr-Ser*-Phe-Gly where Ser* is the phosphorylation site) has similarities to but is distinct from optimal substrates that we previously defined for related basophilic protein kinases such as protein kinase A, Ser/Arg-rich kinases, and protein kinase C family members. The positions most important for high V(max)/K(m) ratio were Arg-3>Arg-5>Arg-7. The substrate specificity of AKT was further investigated by screening a lambdaGEX phage HeLa cell cDNA expression library. All of the substrates identified by this procedure contained Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr) motifs and were in close agreement with the motif identified by peptide library screening. The results of this study should help in prediction of likely AKT substrates from primary sequences.

14-3-3 interacts with regulator of G protein signaling proteins and modulates their activity.

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins (GAPs) that stimulate the inactivation of heterotrimeric G proteins. We have recently shown that RGS proteins may be regulated on a post-translational level (Benzing, T., Brandes, R., Sellin, L., Schermer, B., Lecker, S., Walz, G., and Kim, E. (1999) Nat. Med. 5, 913-918). However, mechanisms controlling the GAP activity of RGS proteins are poorly understood. Here we show that 14-3-3 proteins associate with RGS7 and RGS3. Binding of 14-3-3 is mediated by a conserved phosphoserine located in the Galpha-interacting portion of the RGS domain; interaction with 14-3-3 inhibits the GAP activity of RGS7, depends upon phosphorylation of a conserved residue within the RGS domain, and results in inhibition of GAP function. Collectively, these data indicate that phosphorylation-dependent binding of 14-3-3 may act as molecular switch that controls the GAP activity keeping a substantial fraction of RGS proteins in a dormant state.

MAP kinase pathways activated by stress: the p38 MAPK pathway.

A stress-activated serine/threonine protein kinase, p38 mitogen-activated protein kinase (p38 MAPK), belongs to the MAP kinase superfamily. Diverse extracellular stimuli, including ultraviolet light, irradiation, heat shock, high osmotic stress, proinflammatory cytokines and certain mitogens, trigger a stress-regulated protein kinase cascade culminating in activation of p38 MAPK through phosphorylation on a TGY motif within the kinase activation loop. p38 MAPK appears to play a major role in apoptosis, cytokine production, transcriptional regulation, and cytoskeletal reorganization, and has been causally implicated in sepsis, ischemic heart disease, arthritis, human immunodeficiency virus infection, and Alzheimer's disease. The availability of specific inhibitors helps to clarify the role that p38 MAPK plays in these processes, and may ultimately offer therapeutic benefit for certain critically ill patients.

A novel pro-Arg motif recognized by WW domains.

WW domains mediate protein-protein interactions through binding to short proline-rich sequences. Two distinct sequence motifs, PPXY and PPLP, are recognized by different classes of WW domains, and another class binds to phospho-Ser-Pro sequences. We now describe a novel Pro-Arg sequence motif recognized by a different class of WW domains using data from oriented peptide library screening, expression cloning, and in vitro binding experiments. The prototype member of this group is the WW domain of formin-binding protein 30 (FBP30), a p53-regulated molecule whose WW domains bind to Pro-Arg-rich cellular proteins. This new Pro-Arg sequence motif re-classifies the organization of WW domains based on ligand specificity, and the Pro-Arg class now includes the WW domains of FBP21 and FE65. A structural model is presented which rationalizes the distinct motifs selected by the WW domains of YAP, Pin1, and FBP30. The Pro-Arg motif identified for WW domains often overlaps with SH3 domain motifs within protein sequences, suggesting that the same extended proline-rich sequence could form discrete SH3 or WW domain complexes to transduce distinct cellular signals.