David R. Croucher

Temporal regulation of EGF signalling networks by the scaffold protein Shc1

Cell-surface receptors frequently use scaffold proteins to recruit cytoplasmic targets, but the rationale for this is uncertain. Activated receptor tyrosine kinases, for example, engage scaffolds such as Shc1 that contain phosphotyrosine (pTyr)-binding (PTB) domains. Using quantitative mass spectrometry, here we show that mammalian Shc1 responds to epidermal growth factor (EGF) stimulation through multiple waves of distinct phosphorylation events and protein interactions. After stimulation, Shc1 rapidly binds a group of proteins that activate pro-mitogenic or survival pathways dependent on recruitment of the Grb2 adaptor to Shc1 pTyr sites. Akt-mediated feedback phosphorylation of Shc1 Ser 29 then recruits the Ptpn12 tyrosine phosphatase. This is followed by a sub-network of proteins involved in cytoskeletal reorganization, trafficking and signal termination that binds Shc1 with delayed kinetics, largely through the SgK269 pseudokinase/adaptor protein. Ptpn12 acts as a switch to convert Shc1 from pTyr/Grb2-based signalling to SgK269-mediated pathways that regulate cell invasion and morphogenesis. The Shc1 scaffold therefore directs the temporal flow of signalling information after EGF stimulation.

Tyrosine phosphorylation profiling reveals the signaling network characteristics of basal breast cancer cells

To identify therapeutic targets and prognostic markers for basal breast cancers, breast cancer cell lines were subjected to mass spectrometry-based profiling of protein tyrosine phosphorylation events. This revealed that luminal and basal breast cancer cells exhibit distinct tyrosine phosphorylation signatures that depend on pathway activation as well as protein expression. Basal breast cancer cells are characterized by elevated tyrosine phosphorylation of Met, Lyn, EphA2, epidermal growth factor receptor (EGFR), and FAK, and Src family kinase (SFK) substrates such as p130Cas. SFKs exert a prominent role in these cells, phosphorylating key regulators of adhesion and migration and promoting tyrosine phosphorylation of the receptor tyrosine kinases EGFR and Met. Consistent with these observations, SFK inhibition attenuated cellular proliferation, survival, and motility. Basal breast cancer cell lines exhibited differential responsiveness to small molecule inhibitors of EGFR and Met that correlated with the degree of target phosphorylation, and reflecting kinase coactivation, inhibiting two types of activated network kinase (e.g., EGFR and SFKs) was more effective than single agent approaches. FAK signaling enhanced both proliferation and invasion, and Lyn was identified as a proinvasive component of the network that is associated with a basal phenotype and poor prognosis in patients with breast cancer. These studies highlight multiple kinases and substrates for further evaluation as therapeutic targets and biomarkers. However, they also indicate that patient stratification based on expression/activation of drug targets, coupled with use of multi-kinase inhibitors or combination therapies, may be required for effective treatment of this breast cancer subgroup.

Revisiting the biological roles of PAI2 (SERPINB2) in cancer

Tumour expression of the urokinase plasminogen activator correlates with invasive capacity. Consequently, inhibition of this serine protease by physiological inhibitors should decrease invasion and metastasis. However, of the two main urokinase inhibitors, high tumour levels of the type 1 inhibitor actually promote tumour progression, whereas high levels of the type 2 inhibitor decrease tumour growth and metastasis. We propose that the basis of this apparently paradoxical action of two similar serine protease inhibitors lies in key structural differences controlling interactions with components of the extracellular matrix and endocytosis–signalling co-receptors.

Nedd4 controls animal growth by regulating IGF-1 signaling.

Nedd4 acts through Grb10 to enhance insulin-like growth factor signaling and control animal growth. A Growth-Promoting Ubiquitin Ligase Genetic knockout of the ubiquitin ligase Nedd4 decreases insulin-like growth factor 1 (IGF-1) and insulin signaling and causes delayed embryonic development, reduced growth and body weight, and neonatal lethality. Elevated Grb10 in the Nedd4-deficient cells appears to cause mislocalization of the IGF-1 receptor and prevent receptor signaling at the plasma membrane. Thus, by regulating the abundance of Grb10, a negative regulator of IGF-1 and insulin signaling, Nedd4 positively influences growth. The ubiquitin ligase Nedd4 has been proposed to regulate a number of signaling pathways, but its physiological role in mammals has not been characterized. Here we present an analysis of Nedd4-null mice to show that loss of Nedd4 results in reduced insulin-like growth factor 1 (IGF-1) and insulin signaling, delayed embryonic development, reduced growth and body weight, and neonatal lethality. In mouse embryonic fibroblasts, mitogenic activity was reduced, the abundance of the adaptor protein Grb10 was increased, and the IGF-1 receptor, which is normally present on the plasma membrane, was mislocalized. However, surface expression of IGF-1 receptor was restored in homozygous mutant mouse embryonic fibroblasts after knockdown of Grb10, and Nedd4−/− lethality was rescued by maternal inheritance of a disrupted Grb10 allele. Thus, in vivo, Nedd4 appears to positively control IGF-1 and insulin signaling partly through the regulation of Grb10 function.

Tissue‐type plasminogen activator requires a co‐receptor to enhance NMDA receptor function

Glutamate is the main excitatory neurotransmitter of the CNS. Tissue‐type plasminogen activator (tPA) is recognized as a modulator of glutamatergic neurotransmission. This attribute is exemplified by its ability to potentiate calcium signaling following activation of the glutamate‐binding NMDA receptor (NMDAR). It has been hypothesized that tPA can directly cleave the NR1 subunit of the NMDAR and thereby potentiate NMDA‐induced calcium influx. In contrast, here we show that this increase in NMDAR signaling requires tPA to be proteolytically active, but does not involve cleavage of the NR1 subunit or plasminogen. Rather, we demonstrate that enhancement of NMDAR function by tPA is mediated by a member of the low‐density lipoprotein receptor (LDLR) family. Hence, this study proposes a novel functional relationship between tPA, the NMDAR, a LDLR and an unknown substrate which we suspect to be a serpin. Interestingly, whilst tPA alone failed to cleave NR1, cell‐surface NMDARs did serve as an efficient and discrete proteolytic target for plasmin. Hence, plasmin and tPA can affect the NMDAR via distinct avenues. Altogether, we find that plasmin directly proteolyses the NMDAR whilst tPA functions as an indirect modulator of NMDA‐induced events via LDLR engagement.

Crosstalk and signaling switches in mitogen-activated protein kinase cascades

Mitogen-activated protein kinase (MAPK) cascades control cell fate decisions, such as proliferation, differentiation, and apoptosis by integrating and processing intra- and extracellular cues. However, similar MAPK kinetic profiles can be associated with opposing cellular decisions depending on cell type, signal strength, and dynamics. This implies that signaling by each individual MAPK cascade has to be considered in the context of the entire MAPK network. Here, we develop a dynamic model of feedback and crosstalk for the three major MAPK cascades; extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), c-Jun N-terminal kinase (JNK), and also include input from protein kinase B (AKT) signaling. Focusing on the bistable activation characteristics of the JNK pathway, this model explains how pathway crosstalk harmonizes different MAPK responses resulting in pivotal cell fate decisions. We show that JNK can switch from a transient to sustained activity due to multiple positive feedback loops. Once activated, positive feedback locks JNK in a highly active state and promotes cell death. The switch is modulated by the ERK, p38, and AKT pathways. ERK activation enhances the dual specificity phosphatase (DUSP) mediated dephosphorylation of JNK and shifts the threshold of the apoptotic switch to higher inputs. Activation of p38 restores the threshold by inhibiting ERK activity via the PP1 or PP2A phosphatases. Finally, AKT activation inhibits the JNK positive feedback, thus abrogating the apoptotic switch and allowing only proliferative signaling. Our model facilitates understanding of how cancerous deregulations disturb MAPK signal processing and provides explanations for certain drug resistances. We highlight a critical role of DUSP1 and DUSP2 expression patterns in facilitating the switching of JNK activity and show how oncogene induced ERK hyperactivity prevents the normal apoptotic switch explaining the failure of certain drugs to induce apoptosis.

Activin A is essential for neurogenesis following neurodegeneration.

It has long been proposed that excitotoxicity contributes to nerve cell death in neurodegenerative diseases. Activin A, a member of the transforming growth factor‐β superfamily, is expressed by neurons following excitotoxicity. We show for the first time that this activin A expression is essential for neurogenesis to proceed following neurodegeneration. We found that intraventricular infusion of activin A increased the number of newborn neurons in the dentate gyrus, CA3, and CA1 layers of the normal adult hippocampus and also, following lipopolysaccharide administration, had a potent inhibitory effect on gliosis in vivo and on microglial proliferation in vivo and in vitro. Consistent with the role of activin A in regulating central nervous system inflammation and neurogenesis, intraventricular infusion of follistatin, an activin A antagonist, profoundly impaired neurogenesis and increased the number of microglia and reactive astrocytes following onset of kainic acid‐induced neurodegeneration. These results show that inhibiting endogenous activin A is permissive for a potent underlying inflammatory response to neurodegeneration. We demonstrate that the anti‐inflammatory actions of activin A account for its neurogenic effects following neurodegeneration because co‐administration of nonsteroidal anti‐inflammatory drugs reversed follistatins inhibitory effects on neurogenesis in vivo. Our work indicates that activin A, perhaps working in conjunction with other transforming growth factor‐β superfamily molecules, is essential for neurogenesis in the adult central nervous system following excitotoxic neurodegeneration and suggests that neurons can regulate regeneration by suppressing the inflammatory response, a finding with implications for understanding and treating acute and chronic neurodegenerative diseases. STEM CELLS 2009;27:1330–1346

The Urokinase/PAI-2 Complex A NEW HIGH AFFINITY LIGAND FOR THE ENDOCYTOSIS RECEPTOR LOW DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN

The efficient inactivation of urokinase plasminogen activator (uPA) by plasminogen activator inhibitor type 2 (PAI-2) at the surface of carcinoma cells is followed by rapid endocytosis of the uPA-PAI-2 complex. We now show that one pathway of this receptor-mediated endocytosis is mediated via the low density lipoprotein receptor-related protein (LRP) in prostate cancer cells. Detailed biochemical analyses using ligand binding assays and surface plasmon resonance revealed a novel and distinct interaction mechanism between native, human LRP and uPA-PAI-2. As reported previously for PAI-1, inhibition of uPA by PAI-2 significantly increased the affinity of the complex for LRP (KD of 36 nm for uPA-PAI-2 versus 200 nm for uPA). This interaction was maintained in the presence of uPAR, confirming the validity of this interaction at the cell surface. However, unlike PAI-1, no interaction was observed between LRP and PAI-2 in either the stressed or the relaxed conformation. This suggests that the uPA-PAI-2-LRP interaction is mediated by site(s) within the uPA molecule alone. Thus, as inhibition of uPA by PAI-2 resulted in accelerated clearance of uPA from the cell surface possibly via its increased affinity for LRP, this represents a mechanism through which PAI-2 can clear proteolytic activity from the cell surface. Furthermore, lack of a direct interaction between PAI-2 and LRP implies that downstream signaling events initiated by PAI-1 may not be activated by PAI-2.

Involvement of Lyn and the Atypical Kinase SgK269/PEAK1 in a Basal Breast Cancer Signaling Pathway

Basal breast cancer cells feature high expression of the Src family kinase Lyn that has been implicated in the pathogenicity of this disease. In this study, we identified novel Lyn kinase substrates, the most prominent of which was the atypical kinase SgK269 (PEAK1). In breast cancer cells, SgK269 expression associated with the basal phenotype. In primary breast tumors, SgK269 overexpression was detected in a subset of basal, HER2-positive, and luminal cancers. In immortalized MCF-10A mammary epithelial cells, SgK269 promoted transition to a mesenchymal phenotype and increased cell motility and invasion. Growth of MCF-10A acini in three-dimensional (3D) culture was enhanced upon SgK269 overexpression, which induced an abnormal, multilobular acinar morphology and promoted extracellular signal-regulated kinase (Erk) and Stat3 activation. SgK269 Y635F, mutated at a major Lyn phosphorylation site, did not enhance acinar size or cellular invasion. We show that Y635 represents a Grb2-binding site that promotes both Stat3 and Erk activation in 3D culture. RNA interference-mediated attenuation of SgK269 in basal breast cancer cells promoted acquisition of epithelial characteristics and decreased anchorage-independent growth. Together, our results define a novel signaling pathway in basal breast cancer involving Lyn and SgK269 that offers clinical opportunities for therapeutic intervention.

Signaling pathway models as biomarkers: Patient-specific simulations of JNK activity predict the survival of neuroblastoma patients.

Patient-specific modeling of a cell death–promoting pathway may lead to personalized treatment strategies. Pathway activity as a biomarker Understanding signaling networks may enable prediction of disease mechanisms or responses to therapeutic strategies. Fey et al. showed that constructing a pathway that reproduces the all-or-nothing, switch-like activation of the stress-activated kinase JNK could be used to stratify neuroblastoma patients. Switch-like activation of JNK leads to cell death. By integrating patient-specific information about the abundance of the components of the JNK pathway in neuroblastoma samples into the model, the authors simulated the activity of the pathway and accurately predicted survival on the basis of the dynamic properties of the pathway. Thus, pathway activity served as a biomarker. The results also showed that alterations in the network that prevent the switch-like activation of JNK were associated with poor survival of neuroblastoma patients, thus providing potential molecular mechanisms for the inherent resistance of some of these tumors to treatment. Signaling pathways control cell fate decisions that ultimately determine the behavior of cancer cells. Therefore, the dynamics of pathway activity may contain prognostically relevant information different from that contained in the static nature of other types of biomarkers. To investigate this hypothesis, we characterized the network that regulated stress signaling by the c-Jun N-terminal kinase (JNK) pathway in neuroblastoma cells. We generated an experimentally calibrated and validated computational model of this network and used the model to extract prognostic information from neuroblastoma patient–specific simulations of JNK activation. Switch-like JNK activation mediates cell death by apoptosis. An inability to initiate switch-like JNK activation in the simulations was significantly associated with poor overall survival for patients with neuroblastoma with or without MYCN amplification, indicating that patient-specific simulations of JNK activation could stratify patients. Furthermore, our analysis demonstrated that extracting information about a signaling pathway to develop a prognostically useful model requires understanding of not only components and disease-associated changes in the abundance or activity of the components but also how those changes affect pathway dynamics.