Tilman Brummer

Dual-specificity phosphatases: critical regulators with diverse cellular targets

DUSPs (dual-specificity phosphatases) are a heterogeneous group of protein phosphatases that can dephosphorylate both phosphotyrosine and phosphoserine/phosphothreonine residues within the one substrate. DUSPs have been implicated as major modulators of critical signalling pathways that are dysregulated in various diseases. DUSPs can be divided into six subgroups on the basis of sequence similarity that include slingshots, PRLs (phosphatases of regenerating liver), Cdc14 phosphatases (Cdc is cell division cycle), PTENs (phosphatase and tensin homologues deleted on chromosome 10), myotubularins, MKPs (mitogen-activated protein kinase phosphatases) and atypical DUSPs. Of these subgroups, a great deal of research has focused on the characterization of the MKPs. As their name suggests, MKPs dephosphorylate MAPK (mitogen-activated protein kinase) proteins ERK (extracellular-signal-regulated kinase), JNK (c-Jun N-terminal kinase) and p38 with specificity distinct from that of individual MKP proteins. Atypical DUSPs are mostly of low-molecular-mass and lack the N-terminal CH2 (Cdc25 homology 2) domain common to MKPs. The discovery of most atypical DUSPs has occurred in the last 6 years, which has initiated a large amount of interest in their role and regulation. In the past, atypical DUSPs have generally been grouped together with the MKPs and characterized for their role in MAPK signalling cascades. Indeed, some have been shown to dephosphorylate MAPKs. The current literature hints at the potential of the atypical DUSPs as important signalling regulators, but is crowded with conflicting reports. The present review provides an overview of the DUSP family before focusing on atypical DUSPs, emerging as a group of proteins with vastly diverse substrate specificity and function.

The adaptor protein SLP-65 acts as a tumor suppressor that limits pre-B cell expansion.

Mice deficient in the adaptor protein SLP-65 (also known as BLNK) have reduced numbers of mature B cells, but an increased pre-B cell compartment. We show here that compared to wild-type cells, SLP-65−/− pre-B cells show an enhanced ex vivo proliferative capacity. This proliferation requires interleukin 7 and expression of the pre-B cell receptor (pre-BCR). In addition, SLP-65−/− mice have a high incidence of pre-B cell lymphoma. Reintroduction of SLP-65 into SLP-65−/− pre-B cells led to pre-BCR down-regulation and enhanced differentiation. Our results indicate that SLP-65 regulates a developmental program that promotes differentiation and limits pre-B cell expansion, thereby acting as a tumor suppressor.

Adenosine and cAMP are potent inhibitors of the NF‐κB pathway downstream of immunoreceptors

Anergic B lymphocytes exert compromised signal transduction towards the activation of NF‐κB in response to B cell antigen receptor (BCR) triggering, whereas activation of the ERK pathway appears normal. How this differential down‐regulation of the NF‐κB pathway is regulated remains still elusive. Here, we demonstrate that stimuli known to enhance 3′,5′‐cyclic adenosine monophosphate (cAMP) are capable of selectively suppressing the activation both of NF‐κB downstream of the BCR and Toll‐like receptor 4 in splenic B lymphocytes and of the high‐affinity receptor for IgE in BM‐derived mast cells. This suppression is accomplished by blocking phosphorylation and subsequent degradation of the inhibitor of NF‐κB. A cAMP‐dependent protein kinase (PKA) inhibitor reverses this suppressive effect, indicating that PKA is a downstream effector of cAMP in this process. Importantly, not only drugs that artificially elevate intracellular cAMP levels, but also the nucleoside adenosine, which is known to be a mediator of cellular distress, inhibit the NF‐κB pathway. This suggests that adenosine‐mediated signals represent an important step in the molecular decision process controlling inflammation versus anergic immune responses.

Strong negative feedback from Erk to Raf confers robustness to MAPK signalling

Protein levels within signal transduction pathways vary strongly from cell to cell. Here, we analysed how signalling pathways can still process information quantitatively despite strong heterogeneity in protein levels. We systematically perturbed the protein levels of Erk, the terminal kinase in the MAPK signalling pathway in a panel of human cell lines. We found that the steady‐state phosphorylation of Erk is very robust against perturbations of Erk protein level. Although a multitude of mechanisms exist that may provide robustness against fluctuating protein levels, we found that one single feedback from Erk to Raf‐1 accounts for the observed robustness. Surprisingly, robustness is provided through a fast post‐translational mechanism although variation of Erk levels occurs on a timescale of days.

Function, regulation and pathological roles of the Gab/DOS docking proteins

Since their discovery a little more than a decade ago, the docking proteins of the Gab/DOS family have emerged as important signalling elements in metazoans. Gab/DOS proteins integrate and amplify signals from a wide variety of sources including growth factor, cytokine and antigen receptors as well as cell adhesion molecules. They also contribute to signal diversification by channelling the information from activated receptors into signalling pathways with distinct biological functions. Recent approaches in protein biochemistry and systems biology have revealed that Gab proteins are subject to complex regulation by feed-forward and feedback phosphorylation events as well as protein-protein interactions. Thus, Gab/DOS docking proteins are at the centre of entire signalling subsystems and fulfil an important if not essential role in many physiological processes. Furthermore, aberrant signalling by Gab proteins has been increasingly linked to human diseases from various forms of neoplasia to Alzheimers disease.In this review, we provide a detailed overview of the structure, effector functions, regulation and evolution of the Gab/DOS family. We also summarize recent findings implicating Gab proteins, in particular the Gab2 isoform, in leukaemia, solid tumours and other human diseases.

Increased Proliferation and Altered Growth Factor Dependence of Human Mammary Epithelial Cells Overexpressing the Gab2 Docking Protein

The docking protein Gab2 is a proto-oncogene product that is overexpressed in primary breast cancers. To determine the functional consequences of Gab2 overexpression, we utilized the immortalized human mammary epithelial cell line MCF-10A. In monolayer culture, expression of Gab2 at levels comparable with those detected in human breast cancer cells accelerated epidermal growth factor (EGF)-induced cell cycle progression and was associated with increased basal Stat5 tyrosine phosphorylation and enhanced and/or more sustained EGF-induced Erk and Akt activation. Three-dimensional Matrigel culture of MCF-10A cells resulted in the formation of polarized, growth-arrested acini with hollow lumina. Under these conditions, Gab2 increased cell proliferation during morphogenesis, leading to significantly larger acini, an effect dependent on Gab2 binding to Grb2 and Shp2 and enhanced by recruitment of the p85 subunit of phosphatidylinositol 3-kinase. Pharmacological inhibition of MEK revealed that, in addition to direct activation of phosphatidylinositol 3-kinase, increased Erk signaling also contributed to Gab2-mediated enhancement of acinar size. In addition, Gab2 overcame the proliferative suppression that normally occurs in late stage cultures and conferred independence of the morphogenetic program from exogenous EGF. Finally, higher levels of Gab2 expression led to the formation of large disorganized structures with defective luminal clearance. These findings support a role for Gab2 in mammary tumorigenesis.

Identification of novel ERK-mediated feedback phosphorylation sites at the C-terminus of B-Raf

The extracellular signal-regulated kinase (ERK) pathway plays an important role during the development and activation of B lymphocytes. We have recently shown that B-Raf is a dominant ERK activator in B-cell antigen receptor signalling. We now show that B-Raf is hyperphosphorylated upon BCR engagement and undergoes a prominent electrophoretic mobility shift. This shift correlates with ERK activation and is prevented by the MEK inhibitor U0126. Syk-deficient DT40 B cells display neither dual ERK phosphorylation nor a mobility shift of B-Raf upon BCR engagement. The inducible expression of a constitutively active B-Raf in this mutant line restores dual ERK phosphorylation and the mobility shift of endogenous B-Raf, indicating that these two events are connected to each other. By site-directed mutagenesis studies, we demonstrate that the shift is due to an ERK2-mediated feedback phosphorylation of serine/threonine residues within an evolutionary conserved SPKTP motif at the C-terminus of B-Raf. Replacement of these residues by negatively charged amino acids causes a constitutive mobility shift and a reduction of PC12 cell differentiation. We discuss a model in which ERK-mediated phosphorylation of the SPKTP motif is involved in negative feedback regulation of B-Raf.

Inducible gene deletion reveals different roles for B-Raf and Raf-1 in B-cell antigen receptor signalling

Engagement of the B‐cell antigen receptor (BCR) leads to activation of the Raf–MEK–ERK pathway and Raf kinases play an important role in the modulation of ERK activity. B lymphocytes express two Raf isoforms, Raf‐1 and B‐Raf. Using an inducible deletion system in DT40 cells, the contribution of Raf‐1 and B‐Raf to BCR signalling was dissected. Loss of Raf‐1 has no effect on BCR‐mediated ERK activation, whereas B‐Raf‐deficient DT40 cells display a reduced basal ERK activity as well as a shortened BCR‐mediated ERK activation. The Raf‐1/B‐Raf double deficient DT40 cells show an almost complete block both in ERK activation and in the induction of the immediate early gene products c‐Fos and Egr‐1. In contrast, BCR‐mediated activation of nuclear factor of activated T cells (NFAT) relies predominantly on B‐Raf. Furthermore, complementation of Raf‐1/B‐Raf double deficient cells with various Raf mutants demonstrates a requirement for Ras‐GTP binding in BCR‐mediated activation of both Raf isoforms and also reveals the important role of the S259 residue for the regulation of Raf‐1. Our study shows that BCR‐mediated ERK activation involves a cooperation of both B‐Raf and Raf‐1, which are activated specifically in a temporally distinct manner.

Feedback regulation of lymphocyte signalling.

The development, survival and activation of lymphocytes is controlled by a multitude of extracellular signals in the form of soluble or membrane-bound ligands. Binding of these ligands to receptors on the lymphocyte surface is translated into intracellular signals that are processed in various ways inside the cell and determine its fate. The processing of an incoming signal involves amplification, diversification and termination. Feedback signalling loops have an essential role in the control of these processes, yet our knowledge about these regulatory loops is limited. However, several new feedback regulatory circuits have been recently discovered in lymphocytes and it is probable that more of these circuits will be found in the near future. Here, we give an overview of the present knowledge and working principles of such feedback loops.

Functional characterization of a BRAF insertion mutant associated with pilocytic astrocytoma.

Pilocytic astrocytoma (PA) is emerging as a tumor entity with dysregulated Ras/Raf/MEK/ERK signaling. Common genetic lesions observed in PA, which are linked to aberrant ERK pathway activity, include either NF1 inactivation, KRAS or BRAF gain‐of‐function mutations. To investigate the mutation spectrum within the proto‐oncogene encoding the Ser/Thr‐kinase B‐Raf in more detail, we analyzed 64 primary tumor samples from children with PA including two patients with neurofibromatosis type 1 (NF1). The well‐known BRAFV600E mutation was found in 6/64 (9.38%) of our samples. For the first time, we report concomitant presence of a somatic BRAFV600E mutation in an NF1 patient indicating that more than one Ras/ERK pathway component can be affected in PA. Furthermore, 2/64 (3.13%) of our samples carried a 3‐bp insertion in BRAF resulting in the duplication of threonine 599. This conserved residue is located within the activation segment and, if phosphorylated in a Ras‐dependent manner, plays a key role in Raf activation. Here, we demonstrate that this mutant (B‐RafinsT) and another B‐Raf mutant, which carries two additional threonine residues at this position, display an in vitro kinase activity and cellular MEK/ERK activation potential comparable to those of B‐RafV600E. Notably, replacement of threonines by valine residues had similar effects on B‐Raf activity, suggesting that the distortion of the peptide backbone by additional amino acids rather than the insertion of additional, potential phosphorylation sites destabilizes the inactive conformation of the kinase domain. We also demonstrate that B‐RafinsT and B‐RafV600E, but not B‐Rafwt, provoke drastic morphological alterations in human astrocytes.