Activation In Xenopus Oocytes Research Articles

Ligand with Two Modes of Interaction with the Dopamine D2 Receptor-An Induced-Fit Mechanism of Insurmountable Antagonism.

A solid understanding of the mechanisms governing ligand binding is crucial for rational design of therapeutics targeting the dopamine D2 receptor (D2R). Here, we use G protein-coupled inward rectifier potassium (GIRK) channel activation in Xenopus oocytes to measure the kinetics of D2R antagonism by a series of aripiprazole analogues, as well as the recovery of dopamine (DA) responsivity upon washout. The aripiprazole analogues comprise an orthosteric and a secondary pharmacophore and differ by the length of the saturated carbon linker joining these two pharmacophores. Two compounds containing 3- and 5-carbon linkers allowed for a similar extent of recovery from antagonism in the presence of 1 or 100 μM DA (>25 and >90% of control, respectively), whereas recovery was less prominent (∼20%) upon washout of the 4-carbon linker compound, SV-III-130, both with 1 and 100 μM DA. Prolonging the coincubation time with SV-III-130 further diminished recovery. Curve-shift experiments were consistent with competition between SV-III-130 and DA. Two mutations in the secondary binding pocket (V91A and E95A) of D2R decreased antagonistic potency and increased recovery from SV-III-130 antagonism, whereas a third mutation (L94A) only increased recovery. Our results suggest that the secondary binding pocket influences recovery from inhibition by the studied aripiprazole analogues. We propose a mechanism, supported by in silico modeling, whereby SV-III-130 initially binds reversibly to the D2R, after which the drug-receptor complex undergoes a slow transition to a second ligand-bound state, which is dependent on secondary binding pocket integrity and irreversible during the time frame of our experiments.

The Beta-Arrestin-Biased Dopamine D2 Receptor Ligand, UNC9994, Is a Partial Agonist at G-Protein-Mediated Potassium Channel Activation.

BackgroundPrevious evidence suggests that UNC9994 is a beta-arrestin2-selective agonist at the dopamine D2 receptor, lacking ability both to activate and antagonize G protein-dependent signaling. However, this has only been reported by one laboratory using a single assay.MethodsWe used G protein-coupled inward rectifier potassium channel activation in Xenopus oocytes to investigate UNC9994-induced modulation of G protein-dependent signaling at dopamine D2 receptor and dopamine D3 receptor.ResultsAt dopamine D2 receptor, UNC9994 induced G protein-coupled inward rectifier potassium channel currents that were 15% of the maximal response to dopamine, with an EC50 of 185 nM. At dopamine D3 receptor, the ligand elicited 89% of the maximal dopamine response with an EC50 of 62 nM. Pertussis toxin abolished G protein-coupled inward rectifier potassium channel activation. Furthermore, UNC9994 antagonized dopamine-induced G protein-coupled inward rectifier potassium channel activation at dopamine D2 receptor.ConclusionsUNC9994 modulates G protein-coupled inward rectifier potassium channel channel activation via pertussis toxin-sensitive G proteins at dopamine D2 receptor and dopamine D3 receptor. These findings may have implications for the interpretation of data obtained with this ligand.

Hyperosmotic Shock Engages Two Positive Feedback Loops through Caspase-3-dependent Proteolysis of JNK1-2 and Bid

Hyperosmotic shock induces early calpain activation, Smac/DIABLO release from the mitochondria, and p38/JNK activation in Xenopus oocytes. These pathways regulate late cytochrome c release and caspase-3 activation. Here, we show that JNK1-1 and JNK1-2 are activated early by osmostress, and sustained activation of both isoforms accelerates the apoptotic program. When caspase-3 is activated, JNK1-2 is proteolyzed at Asp-385 increasing the release of cytochrome c and caspase-3 activity, thereby creating a positive feedback loop. Expression of Bcl-xL markedly reduces hyperosmotic shock-induced apoptosis. In contrast, expression of Bid induces rapid caspase-3 activation, even in the absence of osmostress, which is blocked by Bcl-xL co-expression. In these conditions a significant amount of Bid in the cytosol is mono- and bi-ubiquitinated. Caspase-3 activation by hyperosmotic shock induces proteolysis of Bid and mono-ubiquitinated Bid at Asp-52 increasing the release of cytochrome c and caspase-3 activation, and thus creating a second positive feedback loop. Revealing the JNK isoforms and the loops activated by osmostress could help to design better treatments for human diseases caused by perturbations in fluid osmolarity.

Osmostress-induced apoptosis in Xenopus oocytes: role of stress protein kinases, calpains and Smac/DIABLO.

Hyperosmotic shock induces cytochrome c release and capase-3 activation in Xenopus oocytes, but the regulators and signaling pathways involved are not well characterized. Here we show that hyperosmotic shock induces rapid calpain activation and high levels of Smac/DIABLO release from the mitochondria before significant amounts of cytochrome c are released to promote caspase-3 activation. Calpain inhibitors or EGTA microinjection delays osmostress-induced apoptosis, and blockage of Smac/DIABLO with antibodies markedly reduces cytochrome c release and caspase-3 activation. Hyperosmotic shock also activates the p38 and JNK signaling pathways very quickly. Simultaneous inhibition of both p38 and JNK pathways reduces osmostress-induced apoptosis, while sustained activation of these kinases accelerates the release of cytochrome c and caspase-3 activation. Therefore, at least four different pathways early induced by osmostress converge on the mitochondria to trigger apoptosis. Deciphering the mechanisms of hyperosmotic shock-induced apoptosis gives insight for potential treatments of human diseases that are caused by perturbations in fluid osmolarity.

Voltage sensitivities and deactivation kinetics of histamine H3 and H4 receptors

Agonist potency at some neurotransmitter receptors has been shown to be regulated by voltage, a mechanism which has been suggested to play a crucial role in the regulation of neurotransmitter release by inhibitory autoreceptors. Likewise, receptor deactivation rates upon agonist removal have been implicated in autoreceptor function. Using G protein-coupled potassium (GIRK) channel activation in Xenopus oocytes as readout of receptor activity, we have investigated the voltage sensitivities and signaling kinetics of the hH3445 and hH3365 isoforms of the human histamine H3 receptor, which functions as an inhibitory auto- and heteroreceptor in the nervous system. We have also investigated both the human and the mouse homologues of the related histamine H4 receptor, which is expressed mainly on hematopoietic cells. We found that the hH3445 receptor is the most sensitive to voltage, whereas the hH3365 and H4 receptors are less affected. We further observed a marked difference in response deactivation kinetics between the hH3445 and hH3365 isoforms, with the hH3365 isoform being five to six-fold slower than the hH3445 receptor. Finally, using synthetic agonists, we found evidence for agonist-specific voltage sensitivity at the hH4 receptor. The differences in voltage sensitivities and deactivation kinetics between the hH3445, hH3365, and H4 receptors might be relevant to their respective physiological roles.

TRESK Background K+ Channel Is Inhibited by Phosphorylation via Two Distinct Pathways

The two-pore domain K(+) channel, TRESK (TWIK-related spinal cord K(+) channel, KCNK18) is directly regulated by the calcium/calmodulin-dependent phosphatase calcineurin and 14-3-3 adaptor proteins. The calcium signal robustly activates the channel via calcineurin, whereas the anchoring of 14-3-3 interferes with the return of the current to the resting state after the activation in Xenopus oocytes. In the present study, we report that the phosphorylation of TRESK at two distinct regulatory regions, the 14-3-3 binding site (Ser-264) and the cluster of three adjacent serine residues (Ser-274, Ser-276, and Ser-279), are responsible for channel inhibition. The phosphorylation of Ser-264 by protein kinase A accelerated the return of the current of S276E mutant TRESK to the resting state after the calcineurin-dependent activation. In the presence of 14-3-3, the basal current of the S276E mutant was reduced, and its calcineurin-dependent activation was augmented, suggesting that the direct binding of the adaptor protein to TRESK contributed to the basal inhibition of the channel under resting conditions. Unexpectedly, we found that 14-3-3 impeded the recovery of the current of S264E mutant TRESK to the resting state after the calcineurin-dependent activation, despite of the mutated 14-3-3 binding site. This suggests that 14-3-3 inhibited the kinase phosphorylating the regulatory cluster of Ser-274, Ser-276, and Ser-279, independently of the direct interaction between TRESK and 14-3-3. In conclusion, two distinct inhibitory kinase pathways converge on TRESK, and their effect on the calcineurin-dependent regulation is differentially modulated by the functional availability of 14-3-3.

Employing a Mechanistic Model for the Mapk Pathway to Examine the Impact of Cellular all or None Behavior on Overall Tissue Response

The mitogen activated protein kinase (MAPK) cascade is a three-tiered phosphorylation cascade that is ubiquitously expressed among eukaryotic cells. Its primary function is to propagate signals from cell surface receptors to various cytosolic and nuclear targets. Recent studies have demonstrated that the MAPK cascade exhibits an all-or-none response to graded stimuli. This study quantitatively investigates MAPK activation in Xenopus oocytes using both empirical and biologically-based mechanistic models. Empirical models can represent overall tissue MAPK activation in the oocytes. However, these models lack description of key biological processes and therefore give no insight into whether the cellular response occurs in a graded or all-or-none fashion. To examine the propagation of cellular MAPK all-or-none activation to overall tissue response, mechanistic models in conjunction with Monte Carlo simulations are employed. An adequate description of the dose response relationship of MAPK activation in Xenopus oocytes is achieved. Furthermore, application of these mechanistic models revealed that the initial receptor-ligand binding rate contributes to the cells' ability to exhibit an all-or-none MAPK activation response, while downstream activation parameters contribute more to the magnitude of activation. These mechanistic models enable us to identify key biological events which quantitatively impact the shape of the dose response curve, especially at low environmentally relevant doses.

Deciphering the H-Ras pathway in Xenopus oocyte

Xenopus oocytes are arrested in prophase of the first meiotic division. In response to progesterone, they re-enter meiosis and arrest again in metaphase of the second meiotic division. This process, called meiotic maturation, is under the control of the Cyclin B-Cdc2 complex, M phase promoting factor (MPF). Injection of a constitutively active Xenopus H-Ras protein activates MPF, suggesting that Ras proteins could be implicated in the progesterone transduction pathway. The aim of this study was (1) to elucidate the pathway triggered by H-Ras leading to MPF activation in Xenopus oocytes and (2) to investigate whether endogenous H-Ras is involved in the physiological process of meiotic maturation. We generated three constitutively active double mutants, each of them recruiting a single effector in mammalian cells, mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K) or RalGDS. Our results show that the activation of a PI3K-related enzyme is crucial for H-Ras-induced MPF activation, whereas the recruitment of either MAPK or RalGDS is not. However, although the H-Ras/PI3K pathway is functional in Xenopus oocytes, it is not the physiological transducer of progesterone responsible for meiotic resumption.

Remote hot spots mediate protein substrate recognition for the Cdc25 phosphatase

Cdc25B is a phosphatase that catalyzes the dephosphorylation and activation of the cyclin-dependent kinases, thus driving cell cycle progression. We have identified two residues, R488 and Y497, located >20 A from the active site, that mediate protein substrate recognition without affecting activity toward small-molecule substrates. Injection of Cdc25B wild-type but not the R488L or Y497A variants induces germinal vesicle breakdown and cyclin-dependent kinase activation in Xenopus oocytes. The conditional knockout of the cdc25 homolog (mih1) in Saccharomyces cerevisiae can be complemented by the wild type but not by the hot spot variants, indicating that protein substrate recognition by the Cdc25 phosphatases is an essential and evolutionarily conserved feature.

Cytoplasmic Polyadenylation Element (CPE)- and CPE-binding Protein (CPEB)-independent Mechanisms Regulate Early Class Maternal mRNA Translational Activation in Xenopus Oocytes

Meiotic cell cycle progression during vertebrate oocyte maturation requires the correct temporal translation of maternal mRNAs encoding key regulatory proteins. The mechanism by which specific mRNAs are temporally activated is unknown, although both cytoplasmic polyadenylation elements (CPE) within the 3'-untranslated region (3'-UTR) of mRNAs and the CPE-binding protein (CPEB) have been implicated. We report that in progesterone-stimulated Xenopus oocytes, the early cytoplasmic polyadenylation and translational activation of multiple maternal mRNAs occur in a CPE- and CPEB-independent manner. We demonstrate that polyadenylation response elements, originally identified in the 3'-UTR of the mRNA encoding the Mos proto-oncogene, direct CPE- and CPEB-independent polyadenylation of an early class of Xenopus maternal mRNAs. Our findings refute the hypothesis that CPE sequences alone account for the range of temporal inductions of maternal mRNAs observed during Xenopus oocyte maturation. Rather, our data indicate that the sequential action of distinct 3'-UTR-directed translational control mechanisms coordinates the complex temporal patterns and extent of protein synthesis during vertebrate meiotic cell cycle progression.

Distinct regulation of expressed calcium channels 2.3 in Xenopus oocytes by direct or indirect activation of protein kinase C

Protein kinase C (PKC)-dependent regulation of voltage-gated Ca (Ca v; with α 1β1Bα2/δ subunits) channel 2.3 was investigated using phorbol 12-myristate 13-acetate (PMA), or by M 1 muscarinic receptor activation in Xenopus oocytes. The inward Ca 2+-current with Ba 2+ ( I Ba) as the charge carrier was potentiated by PMA or acetyl-β-methylcholine (MCh). The inactivating [ I (inact)] and non-inactivating [ I (noninact)] components of I Ba and the time constant of inactivation τ (inact) were all increased by MCh or PMA. This may be a PKC-dependent action since the effect of MCh and PMA was blocked by Ro-31-8425 or β-pseudosubstrate. MCh effect was blocked by atropine, guanosine-5′- O-(2-thiodiphosphate) trilithium (GDPβS) or U-73122. The effect of MCh but not PMA was blocked by the inhibition of inositol-1,4,5-trisphosphate (IP3) receptors, intracellular Ca 2+ ([Ca 2+] i) or the translocation of conventional PKC (cPKC) with heparin, BAPTA and βC2.4, respectively. While a lower concentration (25 nM) of Ro-31-8425 blocked MCh, a higher concentration (500 nM) of Ro-31-8425 was required to block PMA action. This differential susceptibility of MCh and PMA to heparin, BAPTA, βC2.4 or Ro-31-8425 is suggestive of the involvement of Ca 2+-dependent cPKC in MCh action, whereas cPKC and Ca 2+-independent novel PKC (nPKC) in PMA action. PMA led to additional increase in I Ba that was already potentiated by preadministered MCh (1 or 10 μM), leading to the suggestion that differential phosphorylation sites for cPKC and nPKC may be present in the α 12.3 subunit of Ca v 2.3 channels.

Graded mode of transcriptional induction in yeast pheromone signalling revealed by single-cell analysis.

Signalling pathways typically convert a graded, analogue signal into a binary cellular output. In the several eukaryotic systems that have been investigated to date, including MAP kinase cascade activation in Xenopus oocytes, analogue-to-digital conversion occurs at points in the pathway between receptor activation and the effector mechanism. We used flow cytometry combined with an intracellular fluorescent reporter to examine the characteristics of the yeast pheromone-response pathway. Surprisingly, pheromone response in yeast, which relies on the MAP kinase cascade, behaved in a fundamentally graded manner. Expression of certain exogenous dominant inhibitors of the pathway converted the response to graded-or-none behaviour. These results have implications for the dissection of biological response mechanisms in cells and illustrate how signalling pathways, even homologous ones, may have strikingly different signal propagation/amplification characteristics.

Mos Protein Kinase Activation Requires Hsp90

The protein kinase Mos activates the mitogen-activated protein kinase (MAPK) cascade that controls the meiotic maturation of oocytes. Although the mechanisms regulating the levels of Mos protein have been identified, the mechanisms regulating the kinase activity of Mos are unknown. Fisher, Mandart, and Dorée show that Mos phosphorylation and activation is inhibited in Xenopus oocytes by geldanamycin (GA), an inhibitor of Hsp90, which also blocks MAPK activation in Xenopus oocytes. GA treatment selectively blocked activation of Mos, not its increase in translation in response to hormonal activation of oocyte maturation. Injected mutated Mos, in which the serine that is phosphorylated upon activation is mutated to glutamate to mimic the phosphorylated state, failed to activate MAPK and to induce germinal vesicle breakdown when Hsp90 was inhibited, suggesting that phosphorylation alone was insufficient to activate Mos. Finally, glutathione S-transferase (GST) pull-down experiments were used to demonstrate the physical association of GST-Mos with Hsp70 and Hsp90. The authors suggest that Mos first interacts with Hsp70, then transiently with Hsp90, which leads to activation and reduced binding to Hsp70. Fisher, D.L., Mandart, E., and Dorée, M. (2000) Hsp90 is required for c-Mos activation and biphasic MAP kinase activation in Xenopus oocytes. EMBO J . 19 : 1516-1524. [Abstract] [Full Text]

Hsp90 is required for c-Mos activation and biphasic MAP kinase activation in Xenopus oocytes.

During Xenopus oocyte maturation, the Mos protein kinase is synthesized and activates the MAP kinase cascade. In this report, we demonstrate that the synthesis and activation of Mos are two separable processes. We find that Hsp90 function is required for activation and phosphorylation of Mos and full activation of the MAP kinase cascade. Once Mos is activated, Hsp90 function is no longer required. We show that Mos interacts with both Hsp90 and Hsp70, and that there is an inverse relationship between association of Mos with these two chaperones. We propose that Mos protein kinase is activated by a novel mechanism involving sequential association with Hsp70 and Hsp90 as well as phosphorylation. We also present evidence for a two-phase activation of MAP kinase in Xenopus oocytes.

Are Xenopus oocytes the exception or the rule?

Two universal features of oocyte meiotic maturation are activation of mitogen-activated protein kinase (MAPK) and of maturation-promoting factor (MPF) just prior to germinal vesicle breakdown (GVBD) and entry into the cell cycle. While it is clear that MPF is the pivotal molecule driving cells into M-phase, the role of MAPK in meiosis varies among organisms. In most systems, MAPK is not required for MPF activation. The exception is in Xenopus oocytes, where extensive studies have shown that MAPK is both necessary and sufficient for activating MPF.Fisher and colleagues1xDissociation of MAP kinase activation and MPF activation in hormone-stimulated maturation of Xenopus oocytes. Fisher, D.L. et al. Development. 1999; 126: 4537–4546PubMedSee all References1 recently reported an interesting twist on the MAPK–MPF relationship in Xenopus. Their most exciting finding comes from using a sensitive assay for MAPK activation. They show that MAP kinase is activated several hours before MPF and GVBD, whereas it was accepted previously that MAPK activation just barely precedes MPF activation. They go on to ask, using an Hsp90 inhibitor to prevent MAPK activation indirectly, whether MAPK activation is required for MPF activation. When the inhibitor is added before progesterone stimulation, MAPK is not activated, yet a small percentage of the cells after extended incubation undergo GVBD and activate a low level of MPF. The percentage of cells entering the cell cycle increases if MAPK activity is allowed for a short period just after progesterone stimulation but is then inhibited prior to GVBD, and well in advance of any detectable MPF activity, suggesting that active MAPK does not need to be present in order for MPF to become activated.The somewhat difficult to swallow conclusion drawn from their combination of results is that MAPK is not required for MPF activation in Xenopus oocytes but instead might enhance already activated MPF. This conclusion contradicts extensive data obtained from many other labs, and therefore it is imperative to demonstrate, using other, rigorous methods, that these results are repeatable. In addition, it remains to be explained why, in other species, MPF activation is disconnected completely from MAPK, whereas, in Xenopus, there exists some link between these two molecules.

Dissociation of MAP kinase activation and MPF activation in hormone-stimulated maturation of Xenopus oocytes.

MAP kinase activation occurs during meiotic maturation of oocytes from all animals, but the requirement for MAP kinase activation in reinitiation of meiosis appears to vary between different classes. In particular, it has become accepted that MAP kinase activation is necessary for progesterone-stimulated meiotic maturation of Xenopus oocytes, while this is clearly not the case in other systems. In this paper, we demonstrate that MAP kinase activation in Xenopus oocytes is an early response to progesterone and can be temporally dissociated from MPF activation. We show that MAP kinase activation can be suppressed by treatment with geldanamycin or by overexpression of the MAP kinase phosphatase Pyst1. A transient and low-level early activation of MAP kinase increases the efficiency of cell cycle activation later on, when MAP kinase activity is no longer essential. Many oocytes can still undergo reinitiation of meiosis in the absence of active MAP kinase. Suppression of MAP kinase activation does not affect the formation or activation of Cdc2-cyclin B complexes, but reduces the level of active Cdc2 kinase. We discuss these findings in the context of a universal mechanism for meiotic maturation in oocytes throughout the animal kingdom.

RGS Proteins Determine Signaling Specificity of Gq-coupled Receptors

Regulators of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits, thereby attenuating signaling. RGS4 is a GTPase-activating protein for Gi and Gq class alpha subunits. In the present study, we used knockouts of Gq class genes in mice to evaluate the potency and selectivity of RGS4 in modulating Ca2+ signaling transduced by different Gq-coupled receptors. RGS4 inhibited phospholipase C activity and Ca2+ signaling in a receptor-selective manner in both permeabilized cells and cells dialyzed with RGS4 through a patch pipette. Receptor-dependent inhibition of Ca2+ signaling by RGS4 was observed in acini prepared from the rat and mouse pancreas. The response of mouse pancreatic acini to carbachol was about 4- and 33-fold more sensitive to RGS4 than that of bombesin and cholecystokinin (CCK), respectively. RGS1 and RGS16 were also potent inhibitors of Gq-dependent Ca2+ signaling and acted in a receptor-selective manner. RGS1 showed approximately 1000-fold higher potency in inhibiting carbachol than CCK-dependent signaling. RGS16 was as effective as RGS1 in inhibiting carbachol-dependent signaling but only partially inhibited the response to CCK. By contrast, RGS2 inhibited the response to carbachol and CCK with equal potency. The same pattern of receptor-selective inhibition by RGS4 was observed in acinar cells from wild type and several single and double Gq class knockout mice. Thus, these receptors appear to couple Gq class alpha subunit isotypes equally. Difference in receptor selectivity of RGS proteins action indicates that regulatory specificity is conferred by interaction of RGS proteins with receptor complexes.

Replacement of the H-Ras farnesyl group by lipid analogues: implications for downstream processing and effector activation in Xenopus oocytes.

Ras proteins must undergo a series of posttranslational lipidation steps before they become biologically functional. While the fact that farnesylation is required for subsequent processing steps and indispensable for Ras function has been established, the significance of the isoprenoid structure per se in the context of fully processed Ras is unknown. Here, we describe a novel approach for studying the isoprenoid structure-function relationship in vivo by replacing the H-Ras farnesyl group with synthetic analogues and analyzing their biological functions following microinjection into Xenopus oocytes. We show that the H-Ras farnesyl group can be stripped of most of its isoprenoid features that distinguish it from a fatty acid without any apparent effect on its ability to induce oocyte maturation and activation of mitogen-activated protein kinase. In contrast, replacement by the less hydrophobic isoprenoid geranyl causes severely delayed oocyte activation. Analysis of posttranslational processing reveals a striking correlation between the kinetics of processing, membrane binding, and the onset of biological activity regardless of lipid structure and suggests that slow C-terminal proteolysis and/or methylation can become rate-limiting for H-Ras function. Thus, while our results suggest no stringent requirement for the H-Ras farnesyl structure for effector activation in Xenopus oocytes, they reveal an important role for the lipid present at the farnesylation site in promoting efficient proteolysis and/or methylation which allows rapid palmitoylation, membrane localization, and biological activity. Xenopus oocytes provide a useful in vivo system for the kinetic analysis of the function of the protein of interest present at the physiological dose, which is required for accurate determination of structure-function relationships.

Ras-interacting domain of RGL blocks Ras-dependent signal transduction in Xenopus oocytes

RalGDS family members (RalGDS and RGL) interact with the GTP-bound form of Ras through its effector loop. The C-terminal region (amino acids 602–768) of RGL is responsible for binding to Ras. In this paper we characterized a Ras-interacting domain of RGL using deletion mutants of RGL(602–768). RGL(602–768), RGL(632–768), and RGL(602–734) bound to the GTP-bound form of Ras and inhibited the GAP activity of NF-1. RGL(646–768) showed a low binding activity to Ras and inhibited GAP activity of NF-1 weakly. None of RGL(659–768), RGL(685–768), RGL(602–709), and RGL(602–686) bound to Ras or inhibited GAP activity of NF-1. These results indicate that amino acids 632–734 of RGL constitute a nearly minimal domain that contains the binding element for Ras. RGL(632–734) inhibited v-Ras- but not progesterone-induced Xenopus oocyte maturation. Furthermore, RGL(632–734) inhibited v-Ras- but not v-Raf-dependent extracellular signal-regulated kinase activation in Xenopus oocytes. These results clearly demonstrate that the Ras-interacting domain of RGL is important for Ras-dependent signal transduction in vivo.

Lambda-Interacting Protein, A Novel Protein That Specifically Interacts with the Zinc Finger Domain of the Atypical Protein Kinase C Isotype λ/ι and Stimulates Its Kinase Activity In Vitro and In Vivo

The members of the atypical subfamily of protein kinase C (PKC) show dramatic structural and functional differences from other PKC isotypes. Thus, in contrast to the classical or novel PKCs, they are not activated by diacylglycerol or phorbol esters. However, the atypical PKCs are the target of important lipid second messengers such as ceramide, phosphatidic acid, and 3'-phosphoinositides. The catalytic and pseudosubstrate sequences in the two atypical PKCs (lambda/iota PKC and zeta PKC) are identical but are significantly different from those of conventional or novel PKCs. It has been shown that microinjection of a peptide with the sequence of the pseudosubstrate of the atypical PKC isotypes but not of alpha PKC or epsilon PKC dramatically inhibited maturation and NF-kappa B activation in Xenopus oocytes, as well as reinitiation of DNA synthesis in quiescent mouse fibroblasts. This indicates that either or both atypical isoforms are important in cell signalling. Besides the pseudosubstrate, the major differences in the sequence between lambda/iota PKC and zeta PKC are located in the regulatory domain. Therefore, any functional divergence between the two types of atypical PKCs will presumably reside in that region. We report here the molecular characterization of lambda-interacting protein (LIP), a novel protein that specifically interacts with the zinc finger of lambda/iota PKC but not zeta PKC. We show in this paper that this interaction is detected not only in vitro but also in vivo, that LIP activates lambda/iota PKC but not zeta PKC in vitro and in vivo, and that this interaction is functionally relevant. Thus, expression of LIP leads to the transactivation of a kappa B-dependent promoter in a manner that is dependent on lambda/iota PKC. To our knowledge, this is the first report on the cloning and characterization of a protein activator of a PKC that binds to the zinc finger domain, which has so far been considered a site for binding of lipid modulators. The fact that LIP binds to lambda/iota PKC but not to the highly related zeta PKC isoform suggests that the specificity of the activation of the members of the different PKC subfamilies will most probably be accounted for by proteins like LIP rather than by lipid activators.