Src42 binding activity regulates Drosophila RAF by a novel CNK-dependent derepression mechanism

Connector enhancer of KSR (CNK), an essential component of Drosophila receptor tyrosine kinase/mitogen-activated protein kinase pathways, regulates oppositely RAF function. This bimodal property depends on the N-terminal region of CNK, which integrates RAS activity to stimulate RAF and a bipartite element, called the RAF-inhibitory region (RIR), which binds and inhibits RAF catalytic activity. Here, we show that the repressive effect of the RIR is counteracted by the ability of Src42 to associate, in an RTK-dependent manner, with a conserved region located immediately C-terminal to the RIR. Strikingly, we found that several cnk loss-of-function alleles have mutations clustered in this area and provide evidence that these mutations impair Src42 binding. Surprisingly, the derepressing effect of Src42 does not appear to involve its catalytic function, but critically depends on the ability of its SH3 and SH2 domains to associate with CNK. Together, these findings suggest that the integration of RTK-induced RAS and Src42 signals by CNK as a two-component input is essential for RAF activation in Drosophila.     

Structure of the RAF1-HSP90-CDC37 complex reveals the basis of RAF1 regulation

1. Download : Download high-res image (267KB)
2. Download : Download full-size image

     

RAF protein-serine/threonine kinases: Structure and regulation

A-RAF, B-RAF, and C-RAF are a family of three protein-serine/threonine kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transformation to the cancerous state. RAS mutations occur in 15-30% of all human cancers, and B-RAF mutations occur in 30-60% of melanomas, 30-50% of thyroid cancers, and 5-20% of colorectal cancers. Activation of the RAF kinases requires their interaction with RAS-GTP along with dephosphorylation and also phosphorylation by SRC family protein-tyrosine kinases and other protein-serine/threonine kinases. The formation of unique side-to-side RAF dimers is required for full kinase activity. RAF kinase inhibitors are effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-RAF Val600Glu activating mutation. RAF kinase inhibitors lead to the paradoxical increase in RAF kinase activity in cells containing wild-type B-RAF and wild-type or activated mutant RAS. C-RAF plays a key role in this paradoxical increase in downstream MEK-ERK activation.     

Bimodal regulation of secretion by cytoplasmic Ca2+ as demonstrated by the parathyroid

Bovine parathyroid cells were used to study parathyroid hormone (PTH) release and the cytoplasmic Ca2+ concentration (Cai2+). When the extracellular Ca2+ concentration was decreased from 3.0 to 0.5 mM, perifused cells reacted with rapid stimulation of PTH release. However, a further reduction of extracellular Ca2+ to less than 10 nM resulted in prompt inhibition. Both effects were readily reversible. Using the intracellular Ca2+ indicator quin-2 also as a buffer for calcium it was possible to control Cai2+ within the 20-600 nM range. PTH release was found to increase with Cai2+ up to 200 nM but was gradually suppressed above this concentration.     

Cooperative regulation of the cell division cycle by the protein kinases RAF and AKT

The RAS-activated RAF-->MEK-->extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3'-kinase)-->PDK1-->AKT signaling pathways are believed to cooperate to promote the proliferation of normal cells and the aberrant proliferation of cancer cells. To explore the mechanisms that underlie such cooperation, we have derived cells harboring conditionally active, steroid hormone-regulated forms of RAF and AKT. These cells permit the assessment of the biological and biochemical effects of activation of these protein kinases either alone or in combination with one another. Under conditions where activation of neither RAF nor AKT alone promoted S-phase progression, coactivation of both kinases elicited a robust proliferative response. Moreover, under conditions where high-level activation of RAF induced G(1) cell cycle arrest, activation of AKT bypassed the arrest and promoted S-phase progression. At the level of the cell cycle machinery, RAF and AKT cooperated to induce cyclin D1 and repress p27(Kip1) expression. Repression of p27(Kip1) was accompanied by a dramatic reduction in KIP1 mRNA and was observed in primary mouse embryo fibroblasts derived from mice either lacking SKP2 or expressing a T187A mutated form of p27(Kip1). Consistent with these observations, pharmacological inhibition of MEK or PI3'-kinase inhibited the effects of activated RAS on the expression of p27(Kip1) in NIH 3T3 fibroblasts and in a panel of bona fide human pancreatic cancer cell lines. Furthermore, we demonstrated that AKT activation led to sustained activation of cyclin/cdk2 complexes that occurred concomitantly with the removal of RAF-induced p21(Cip1) from cyclin E/cdk2 complexes. Cumulatively, these data strongly suggest that the RAF-->MEK-->ERK and PI3'K-->PDK-->AKT signaling pathways can cooperate to promote G(0)-->G(1)-->S-phase cell cycle progression in both normal and cancer cells.     

Bimodal regulation of an Elk subfamily K+ channel by phosphatidylinositol 4,5-bisphosphate

Phosphatidylinositol 4,5-bisphosphate (PIP₂) regulates Shaker K⁺ channels and voltage-gated Ca²⁺ channels in a bimodal fashion by inhibiting voltage activation while stabilizing open channels. Bimodal regulation is conserved in hyperpolarization-activated cyclic nucleotide–gated (HCN) channels, but voltage activation is enhanced while the open channel state is destabilized. The proposed sites of PIP₂ regulation in these channels include the voltage-sensor domain (VSD) and conserved regions of the proximal cytoplasmic C terminus. Relatively little is known about PIP₂ regulation of Ether-á-go-go (EAG) channels, a metazoan-specific family of K⁺ channels that includes three gene subfamilies, Eag (Kv10), Erg (Kv11), and Elk (Kv12). We examined PIP₂ regulation of the Elk subfamily potassium channel human Elk1 to determine whether bimodal regulation is conserved within the EAG K⁺ channel family. Open-state stabilization by PIP₂ has been observed in human Erg1, but the proposed site of regulation in the distal C terminus is not conserved among EAG family channels. We show that PIP₂ strongly inhibits voltage activation of Elk1 but also stabilizes the open state. This stabilization produces slow deactivation and a mode shift in voltage gating after activation. However, removal of PIP₂ has the net effect of enhancing Elk1 activation. R347 in the linker between the VSD and pore (S4–S5 linker) and R479 near the S6 activation gate are required for PIP₂ to inhibit voltage activation. The ability of PIP₂ to stabilize the open state also requires these residues, suggesting an overlap in sites central to the opposing effects of PIP₂ on channel gating. Open-state stabilization in Elk1 requires the N-terminal eag domain (PAS domain + Cap), and PIP₂-dependent stabilization is enhanced by a conserved basic residue (K5) in the Cap. Our data shows that PIP₂ can bimodally regulate voltage gating in EAG family channels, as has been proposed for Shaker and HCN channels. PIP₂ regulation appears fundamentally different for Elk and KCNQ channels, suggesting that, although both channel types can regulate action potential threshold in neurons, they are not functionally redundant.     

Polar expression of ErbB-2/HER2 in epithelia. Bimodal regulation by Lin-7

ErbB-2/HER2 drives epithelial malignancies by forming heterodimers with growth factor receptors. The primordial invertebrate receptor is sorted to the basolateral epithelial surface by binding of the PDZ domain of Lin-7 to the receptor's tail. We show that all four human ErbBs are basolaterally expressed, even when the tail motif is absent. Mutagenesis of hLin-7 unveiled a second domain, KID, that binds to the kinase region of ErbBs. The PDZ interaction mediates stabilization of ErbB-2 at the basolateral surface. On the other hand, binding of KID is involved in initial delivery to the basolateral surface, and in its absence, unprocessed ErbB-2 molecules are diverted to the apical surface. Hence, distinct domains of Lin-7 regulate receptor delivery to and maintenance at the basolateral surface of epithelia.     

Mechanisms of translational regulation in Drosophila

Translational regulation plays an essential role in many phases of the Drosophila life cycle. During embryogenesis, specification of the developing body pattern requires co-ordination of the translation of oskar, gurken and nanos mRNAs with their subcellular localization. In addition, dosage compensation is controlled by Sex-lethal-mediated translational regulation while dFMR1 (the Drosophila homologue of the fragile X mental retardation protein) controls translation of various mRNAs which function in the nervous system. Here we describe some of the mechanisms that are utilized to regulate these various processes. Our review highlights the complexity that can be involved with multiple factors employing different mechanisms to control the translation of a single mRNA.     

Stem cell regulation by JAK/STAT signaling in Drosophila

Stem cells have become one of the "buzz" topics in the last decade or so. One of the best systems to study adult stem cells in vivo is in the model organism, Drosophila melanogaster. One hundred years of genetic analysis, a sequenced and highly annotated genome and genomics makes this a difficult organism to avoid. The JAK/STAT pathway has been shown to regulate stem cells during haematopoiesis and gametogenesis in Drosophila. In this review we cover the current literature and contrast each group of stem cells with respect to JAK/STAT signaling.     

Bimodal regulation of the human H1 histamine receptor by G protein-coupled receptor kinase 2

The H1 histamine receptor (H1HR) is a member of the G protein-coupled receptor superfamily and regulates numerous cellular functions through its activation of the G(q/11) subfamily of heterotrimeric G proteins. Although the H1HR has been shown to undergo desensitization in multiple cell types, the mechanisms underlying the regulation of H1HR signaling are poorly defined. To address this issue, we examined the effects of wild type and mutant G protein-coupled receptor kinases (GRKs) on the phosphorylation and signaling of human H1HR in HEK293 cells. Overexpression of GRK2 promoted H1HR phosphorylation in intact HEK293 cells and completely inhibited inositol phosphate production stimulated by H1HR, whereas GRK5 and GRK6 had lesser effects on H1HR phosphorylation and signaling. Interestingly, catalytically inactive GRK2 (GRK2-K220R) also significantly attenuated H1HR-mediated inositol phosphate production, as did an N-terminal fragment of GRK2 previously characterized as a regulator of G protein signaling (RGS) protein for Galpha(q/11). Disruption of this RGS function in holo-GRK2 by mutation (GRK2-D110A) partially reversed the quenching effect of GRK2, whereas deletion of both the kinase activity and RGS function (GRK2-D110A/K220R) effectively relieved the inhibition of inositol phosphate generation. To evaluate the role of endogenous GRKs on H1HR regulation, we used small interfering RNAs to selectively target GRK2 and GRK5, two of the primary GRKs expressed in HEK293 cells. A GRK2-specific small interfering RNA effectively reduced GRK2 expression and resulted in a significant increase in histamine-promoted calcium flux. In contrast, knockdown of GRK5 expression was without effect on H1HR signaling. These findings demonstrate that GRK2 is the principal kinase mediating H1 histamine receptor desensitization in HEK293 cells and suggest that rapid termination of H1HR signaling is mediated by both the kinase activity and RGS function of GRK2.     

Differences in regulation of Drosophila and vertebrate integrin affinity by talin

Integrin-mediated cell adhesion is essential for development of multicellular organisms. In worms, flies, and vertebrates, talin forms a physical link between integrin cytoplasmic domains and the actin cytoskeleton. Loss of either integrins or talin leads to similar phenotypes. In vertebrates, talin is also a key regulator of integrin affinity. We used a ligand-mimetic Fab fragment, TWOW-1, to assess talin's role in regulating Drosophila alphaPS2betaPS affinity. Depletion of cellular metabolic energy reduced TWOW-1 binding, suggesting alphaPS2betaPS affinity is an active process as it is for vertebrate integrins. In contrast to vertebrate integrins, neither talin knockdown by RNA interference nor talin head overexpression had a significant effect on TWOW-1 binding. Furthermore, replacement of the transmembrane or talin-binding cytoplasmic domains of alphaPS2betaPS with those of human alphaIIbbeta3 failed to enable talin regulation of TWOW-1 binding. However, substitution of the extracellular and transmembrane domains of alphaPS2betaPS with those of alphaIIbbeta3 resulted in a constitutively active integrin whose affinity was reduced by talin knockdown. Furthermore, wild-type alphaIIbbeta3 was activated by overexpression of Drosophila talin head domain. Thus, despite evolutionary conservation of talin's integrin/cytoskeleton linkage function, talin is not sufficient to regulate Drosophila alphaPS2betaPS affinity because of structural features inherent in the alphaPS2betaPS extracellular and/or transmembrane domains.     

Transcriptional regulation of the Drosophila raf proto-oncogene by Drosophila STAT during development and in immune response

The Drosophila raf (D-raf) gene promoter contains a recognition consensus sequence for Drosophila STAT (D-STAT). By band mobility shift assay, we detected a factor binding to the D-STAT-recognition sequence in extracts of cultured Drosophila cells treated with vanadate peroxide. UV-cross-linking analyses suggested the size of the binding factor to be almost same as that of D-STAT. Furthermore, the binding activity was increased in cells cotransfected with HOP and D-STAT expression plasmids. These results strongly suggest that D-STAT binds to the D-STAT recognition sequence in the D-raf gene promoter. Transient luciferase expression assay using Schneider 2 cells indicated that the D-raf gene promoter is activated by D-STAT through the D-STAT-binding site. Furthermore, analyses with transgenic flies carrying Draf-lacZ fusion genes with and without mutations in the D-STAT-binding site pointed to an important role in D-raf gene promoter activity throughout development. We also found that the D-STAT-binding site is required for injury-induced activation of the D-raf gene promoter. Here we propose that D-STAT can participate in regulation of the mitogen-activated protein kinase cascade through D-raf gene activation.     

Female-specific regulation of cuticular hydrocarbon biosynthesis by dopamine in Drosophila melanogaster

The role of dopamine (DA) is investigated in cuticular hydrocarbon biosynthesis in Drosophila melanogaster with three different approaches: use of DA-deficient mutants (dopa decarboxylase temperature sensitive mutants reared at restrictive temperature, and rescued by dopamine ingestion or by pale mutants partially rescued by a tyrosine hydroxylase construction), pharmacological treatments (tyrosine hydroxylase inhibitors) and topical application on decapitated flies. We report that DA specifically regulates diene hydrocarbon biosynthesis, which is female specific. Our results suggest that DA acts in adult flies within the first hours of imaginal life and that DA production from the brain is crucial for this process. Thus, DA contributes to reproduction in D. melanogaster by acting during a critical period during development of young adults.