Principles of Cell Signaling and Biological Consequences


Principles of Cell Signaling and Biological Consequences

Journal Club Discussion of Cell 116, 855-967 (2004)

Jan 31 2005 8:52AM

A. Chan, R. Iyengar, S. Aaronson, A. Caplan, S. Salton, and M. M. Zhou

Students should read the following article, the discussion lead-in summary, and respond to this topic on the questions listed at the end of the summary:

P.T. Wan, M. J. Garnett, S. M. Roe, S. Lee, D. Niculescu-Duvaz, V. M. Good, C. M. Jones, C. J. Marshall, C. J. Springer, D. Barford, R. Marais, Cancer Genome Project, Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 116, 855-867 (2004).

Discussion Lead-In Summary

This article illustrates the important concept that the mammalian mitogen-activated protein kinase (MAPK) pathway is a major target for oncogenic activations in human cancer. It was known for nearly two decades that the human N-RAS oncogene is mutated in approximately 20% of human melanomas. The most frequent mutations reside in codon 61, with these lesions found commonly in sun-exposed surfaces. However, the genetic alterations that could account for the remaining melanomas lacking N-RAS mutations had eluded researchers. Clues to this conundrum were provided by Wan et al. (also by Davies et. al. Nature 417, 949-954, 2002) in which B-Raf was identified to be the culprit. Cumulative data from several laboratories have found B-Raf to be mutated to as high as 70% in melanocytic nevi and vertical growth phase melanomas. Intriguingly, unlike N-Ras tumors, these melanomas do not appear to be associated with an etiology involving UV-exposure.

Analyzing the sites of mutation reveal that they are clustered into two major hotspots. These regions represent catalytic motifs that are conserved among various kinases and are absolutely essential for signal transduction. The first mutation cluster accounts for over 90% of all B-Raf mutations in melanomas. It is located at codon Val 599 in the activation segment that spans codon 582-622. Within this 20 amino acid region lies two phosphorylation sites at Thr 598 and Ser 602. In addition, Asp 593 of the conserved DFG motif serves to stabilize an Mg2+ ion important for phosphotransfer. In the inactive state of the B-Raf kinase, Val 599 interacts with the P-loop, causing the DFG motif to attain a conformation that is not amenable for catalysis. Thus, a Val to Glu mutation at codon 599 in melanomas is predicted to release this constrain such that Asp 593 can now direct the transfer of the phosphate to the B-Raf substrate, MEK.

The second finding of this paper illustrates a well-known feature of the MAPK pathway and that is the ability to form signaling complexes. In a very low fraction of melanomas, B-Raf acquires mutations in the P-loop, which is responsible for binding to the phosphate moieties of ATP. The primary amino acid sequence is characterized by the Gly-X-Gly-X-X-Gly motif found in majority of mammalian kinases. Mutations at one of the glycines have the paradoxical effect of reducing the kinase activity of the resulting mutant B-Raf protein. This led the authors to propose a novel mechanism that can explain the oncogenicity of these mutants. It is believed that these kinase-impaired B-Raf proteins first heterodimerize with c-Raf. This induces a conformational change in c-Raf leading to its activation. c-Raf activated through this novel mechanism will in turn stimulate MEK and then ERK. These data raise several intriguing questions.

  • What is the physiological relevance of B-Raf/c-Raf complexes in MAPK signaling?
  • Is B-Raf/c-Raf complex only formed in response to a certain upstream signals?

Discussion Questions and Instructions

Jan 31 2005 9:08AM

Andrew Chan

Discussion questions are divided into two groups (Group A and Group B). Students should select one set of questions within each group to discuss. Students may search PubMed in order to answer the questions, as well as rely on materials presented in class. Answers should be as concise as possible. Because there may not be "model" answers to all of the questions posed, please feel free to submit creative responses. Include in your response the citation to any articles that support your answers.

This discussion forum will be open for students of the Cell Signaling Systems course at Mount Sinai School of Medicine, New York, NY until 15 February 2005. If you are a student in the Cell Signaling Systems course at Mount Sinai, please do not submit your response anonymously, so that the instructors attribute your responses to you.

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Group A: Discussion Question 1

Jan 31 2005 9:10AM

Andrew Chan

Soluble mitogens, such as lysophosphatidic acid (LPA) and epidermal growth factor (EGF), can both stimulate mitogen-activated protein kinase (MAPK) and proliferation in cultured cells. However, the receptors for these two growth factors are vastly different. Describe the distinct features of these two classes of receptors and their downstream signaling molecules leading to the activation of MAPK.

Group A: Discussion Question 2

Jan 31 2005 9:13AM

Andrew Chan

The Ras family of GTP-binding proteins are molecular switches, transmitting a wide array of extracellular signals. Describe the molecular events involved in regulating the ON-OFF states of G proteins. Name two regulators of Ras that can control the equilibrium of this switch. Also, identify two genetic changes in cancer cells that have been shown to cause the constitutive activation of Ras.

Group A: Discussion Question 3

Jan 31 2005 9:15AM

Andrew Chan

Protein phosphorylation is a critical biochemical reaction in intracellular signaling and it is catalyzed by protein kinases. Constitutive activation of protein kinases can lead to oncogenicity as have been described for Bcr-Abl and B-Raf. (i) Name two molecular events that commonly occur in protein kinases that are necessary for full activation. (ii) Name two major domains within the kinase catalytic pocket and describe their roles in catalyzing the phosphotransfer reaction.

Group B: Discussion Question 1

Jan 31 2005 9:17AM

Andrew Chan

The MAPK kinase pathway is composed of several classes of signaling molecules, such as receptors, adaptors, G proteins, phosphatases, and various kinases. One commonly used research tool for unraveling the biological functions of signaling molecules is dominant-negative (DN) mutants. Select a signaling molecule in this pathway and (i) describe how you would construct a dominant-negative mutant, (ii) explain how it works as a DN mutant, and (iii) describe some of the pitfalls in using that particular mutant.

Group B: Discussion Question 2

Jan 31 2005 9:20AM

Andrew Chan

The kinetics of MAPK activation plays an important role in determining the biological outcomes of upstream signals. For example, although epidermal growth factor (EGF) stimulates a transient activation of MAPK, nerve growth factor (NGF) triggers a more sustained stimulation. Speculate on the molecular mechanisms involved in causing this divergence in signaling. Also, what are your thoughts on how this difference in MAPK activation can lead to such drastic differences in biological outcomes - proliferation verse differentiation?

Group B: Discussion Question 3

Jan 31 2005 9:22AM

Andrew Chan

The small molecule compound, BAY43-9006, is a promising drug for treating certain forms of human cancer. Describe the major mechanism responsible for its inhibitory action. Like another kinase inhibitors, STI-571 (Gleevec), it is possible that tumors will eventually develop resistance. Name two plausible changes in cancer cells that might account for the loss of the effectiveness of these kinase inhibitors. Describe a strategy to overcome those particular forms of drug resistance.

Final Forum

Apr 25 2005 2:01PM

Course Directors

Functional Consequences of Interactions between Signaling Pathways

A general theme that has emerged from this course is that interactions between signaling pathways (cross-talk) have substantial functional consequences.

1) Choose a recent (within the past two years) primary publication that you think demonstrates the functional consequences of cross-talk and write a brief (200-500 word) description of why you think the experiments in this paper highlight the functional consequences of cross talk.

Log into Science's STKE with your username and password. Then use the "Respond to this message" link in the left navigation area to submit your description and the full reference to the selected primary publication in this Final Forum thread in the discussion by 10 May 2005.

Students should enter their assigned identification number, as the first and last name entries for the Forum submission form. Please enter your correct email address so that the editors can contact you if there is a problem with your submission. Your identification number is known only to the Course Directors and your email address will not show with the submission, thereby maintaining your privacy. Do not check the anonymous submission box or your identification number will not show.

2) Please read all of the descriptions posted by your fellow students under the Final Forum discussion thread, as well as the abstracts of the papers cited. Select two of the papers and descriptions, excluding your own, for further comment. Write a commentary consisting of two 200-300 word paragraphs to submit to the Final Forum using your identification number as described above.

The first paragraph should focus on your evaluation of whether the publication convincingly demonstrates functional consequences of interactions between signaling pathways.

The second paragraph should comment on the description written by the student who selected the paper. State why you agree or disagree with the description, and if additional arguments in support or criticisms against the study need to be made.

Comments in both paragraphs should be supported with additional references to primary publications or review articles.

Post your commentary on the appropriate second-level thread in the Final Forum discussion thread by 20 May 2005.

Final Forum: Student #4

May 10 2005 7:34AM

Noura S. Abul-Husn

Selected Publication:

Li Y, Eitan S, Wu J, Evans CJ, Kieffer B, Sun X, Polakiewicz RD.2003. Morphine induces desensitization of insulin receptorsignaling. Mol Cell Biol. 23(17): 6255-6266

There is increasing evidence that a number of G-protein coupledreceptors (GPCRs) functionally interact with receptor tyrosinekinases (RTKs). The notion that such cross-talk could occurbetween the mu-opioid receptor (MOR) and the insulin receptor(IR) stemmed from earlier observations that centrally administeredmorphine resulted in hyperglycemic effects (Feldberg & Gupta,1974), and that insulin increased the intrinsic efficacy of MORagonists in Xenopus oocytes (McLaughlin et al., 2001). The MORand the IR activate similar signaling pathways, including the Aktand ERK pathways. Previous studies by Polakiewicz et al. (1998)established a functional role for MOR-induced ERK activation in afeedback signal required for MOR desensitization. The findings inthe present paper by Li et al. (2003) indicate that MOR-inducedERK activation can also play a functional role in modulatingdownstream IR signaling pathways.

In cells expressing endogenous or transfected MOR, IR, andinsulin receptor substrate IRS-1, morphine pretreatmentcompletely abolished insulin-induced phosphorylation of Akt andERK in a receptor-specific and ERK-dependent manner. Theeffects of morphine on serine/threonine phosphorylation of the IRand IRS-1 were investigated, since such phosphorylation eventsattenuate insulin signaling and have been implicated in insulinresistance (Zick, 2001). As predicted, morphine induced serinephosphorylation of the IR and IRS-1, in an ERK-dependentmanner. The effect of morphine on the integrity of IR/IRS-1/effectorcomplexes was then examined. Morphine attenuated the insulin- mediated tyrosine phosphorylation of the adaptor protein Shc,impairing the formation of an active complex among the IR, Shc,and Grb2. This could explain the mechanism for morphine- induced attenuation of IR signaling to ERK, as Shc normallyinteracts with Grb2 to relay the insulin signal to the Ras/ERKcascade. Furthermore, morphine disrupted the interaction betweenIRS-1 and the p85 subunit of phosphatidylinositol 3-kinase (PI3K)by reducing tyrosine phosphorylation of IRS-1 at its p85 bindingsite. This could explain the mechanism for morphine-inducedattenuation of IR signaling to Akt, since PI3K activation leads tostimulation of Akt. Thus, MOR activation of the ERK cascadeappears to attenuate IR signaling to Akt and ERK by disrupting theintegrity of two different signaling complexes. Finally, acutemorphine administration increased IRS-1 serine phosphorylationin discrete brain regions of wild-type but not MOR knockout mice,illustrating the receptor specificity and physiological relevance ofthese findings.

Overall, this paper convincingly demonstrates the functional cross- talk between the MOR and the IR. A series of well-designedexperiments were performed in both heterologous cell lines andnative expression systems, using a combination ofpharmacological, biochemical, and genetic tools to provide directevidence for unidirectional cross-talk between MOR and IRsignaling pathways. The importance of signaling complexes insignal transduction was also illustrated, as the disruption of thesecomplexes by an independent signaling cascade can completelyalter the outcome of the signaling pathway.

The cross-talk between the MOR and the IR could explainmorphine-induced insulin resistance. As this has not beenexplicitly established, however, it will be necessary to furtherexplore the direct interactions between morphine and insulinsignaling systems to determine whether the hyperglycemic effectsof morphine can truly be explained by cross-talk.


Feldberg W & Gupta KP. 1974. Morphine hyperglycemia. JPhysiol. 238: 487-502 [PubMed]

McLaughlin JP & Chavkin C. 2001. Tyrosine phosphorylation ofthe mu-opioid receptor regulates agonist intrinsic efficacy. MolPharmacol. 59: 1360-1368 [PubMed] [Online Journal]

Polakiewicz RD et al. 1998. A mitogen-activated protein kinasepathway is required for mu-opioid receptor desensitization. J BiolChem. 273: 12402-12406 [PubMed] [Online Journal]

Zick Y. 2001. Insulin resistance: a phosphorylation-baseduncoupling of insulin signaling. Trends Cell Biol. 11: 437-441 [PubMed] [Online Journal]

Student #7 Response to Student #4

May 19 2005 6:46AM

Student7 Student7

This paper deals with cross-talk between GPCRs and RTKs, specifically looking at MOR and IR signaling. There was evidence of this cross-talkpreviously, and this paper demonstrates it in more detail. Treating cellswith morphine reduced IR signaling to the Akt and ERK pathways. Thiseffect was specific, as it could be inhibited by a MOR antagonist or MEKinhibitors. Using an antibody the authors generated, they was thatmorphine induced serine phosphorylation of the IR, and reduced theassociation of the IR with Shc. This disrupted formation of the IR, Shc,GRB2 signaling complex. Morphine also decreased tyrosine phosphorylationof IR at the binding sites for p85, leading to a disruption of thiscomplex and PI3K signaling (p85 is the regulatory subunit of PI3K). Thus,MORs can affect IR signaling by disrupting 2 different signalingcomplexes.

I agree with student's assessment of the paper, that itdemonstrates functional cross-talk between the 2 signaling pathways. It is important that the authors demonstrated their results in heterologous andnative cell systems, as well as showing that it may be specific to certain areas of the brain. I also agree that the paper reinforces the importanceof the formation of signaling complexes and illustrates how an independent pathway (MOR) can disrupt complexes important to another pathway (IR),thus changing the signaling ability of that pathway.

Student #6 Response to Student #4

May 20 2005 10:55AM

#6 student

Morphine, acts principally on the mu-opioid receptor (MOR), has beenshown to influence glucose homeostasis which is mainly regulated byinsulin. The studies in this paper examine the mechanism underlying theperturbation resulted from morphine on the insulin receptor (IR) mediatedsignaling pathways. It showed acute morphine treatment causes 1) serinephosphorylation of the IR and impaired the formation of signaling complexamong IR, Shc and Grb2, and 2) serine 612 phosphorylation in IRS-1resulting in reduced tyrosine phosphorylation YMXM p85-binding motifs, and a weakening in the association of IRS-1/pp85 phosphotidylinositol 3-kinase complex. The latter finding was not observed in chronic treatment. Theauthors suggested that these interactions are likely to be where crosstalk occurs. These studies provided insights into how morphine, upon binding to its receptor can attenuate the IR signaling and that such interactions may lead to the perturbance in glucose homeostasis observed. This studydemonstrates an independent signaling cascade can influence the signalingactivities of a second independent pathway in a unidirectional manner.

I agree with the comments of Student#4 in regards to the insightsgained from this study in examining the underlying mechanism of howmorphine influence IR mediated pathway and that the paper illustrate aunidirectional crosstalk between the two pathways. More experiments are needed to confirm the importance of the interactionsobserved and their importance in influencing the IR activities in thecontext of glucose homeostasis. It will also be interesting to furtherexplore why chronic treatment with morphine do not affect the associationof IRS-1/Grb2 complex and if there is possibility of IR pathway "overriding" the initial perturbance posed by the acute morphinetreatment in chronic treatment.

Student #14 Response

May 20 2005 12:57PM

14 14

This paper investigates the crosstalk between the Mu opioid receptorpathway and the insulin receptor pathway, which is an example of aninteraction between GPCR and RTK pathwayw. The model is that the MORpathway is a negative modulator of IR-mediated activation of the Act andERK cascades. The activation of the MOR pathway caused the loss of IR- dependent phosphorylation of Act and ERK, and MOR activation was shown todisrupt the IRS-1/p85 PI3K and the IR/Shc/Grb2 complexes, which are needed for IR signal transduction. Finally, they demonstrate that treatment with morphine induces phosphorylation of IRS-1 in certain brain regions of wild -type mice, but this effect is abolished in MOR knockout mice. Thecombination of biochemical and mouse studies reinforces the authors'case; the experiments with MOR-knockout mice produced especially strongevidence of the in-vivo role of this crosstalk.

I feel that the results presented by the authors are convincing; Iagree with the conclusions of this paper, and with Student #8'sassessment thereof. I especially liked the fact that the authorsmentioned the physiological background of their work, i.e. the role ofopiods in glucose homeostasis. With the epidemic of Type II diabetessweeping across our nation, any insights into the desensitization toinsulin receptor signaling take on a high degree of medical importance. However, one of my concerns is that the biochemical work was done in cells overexpressing various signaling pathway components, which is a ratherartificial system. It would have been a valuable additional experiment to use siRNA to knock out various pathway components in cells which expressthem endogenously. The only other issue is that I would have liked forthe authors to include a diagram of their model. This would make thepaper much more clear, especially since the authors propose that the MORpathway disrupts the IRS-1/p85 PI3K and IR/Shc/Grb2 signaling complexesformed by the IR pathway, but not the IRS-1/Grb2 complex. Such complexinteractions are best demonstrated with diagrams. Aside from this, Ifound this paper clear and well-presented.

Final Forum: Student #6

May 10 2005 7:33AM

Student 6 Student 6

Hasson et al. EGFR signaling attenuates Groucho-dependent repressionto antagonize Notch transcriptional Notch transcriptional output. 2005Nature Genetics pp101-105. [PubMed] [Online Journal]

Antagonism between EGFR and Notch pathways is well documented but the level/junction at which crosstalk occurs has yet been fully understood.Price et al, in 1997, reported genetic interactions between Groucho (Gro)and signaling components of EGFR pathways, and postulated that Gro, beinga co-repressor may be involved in the antagonistic interaction betweenEGFR and Notch signaling. In the paper highlighted, Hasson et al, usingboth biochemical procedures and mutagenesis, showed that Gro, indeed,serves as a junction for the interaction between EGFR and Notch pathways.In addition, they showed that in response to MAPK, phosphorylation of Groleads to a decrease in Gro repressing capacity. With prior knowledge thatEGFR pathway antagonizes Notch pathways in wing patterning and that Groand its repressor partner, Notch effector E(spl)mbeta, are antiveindeterminants while EGFR pathway promotes vein formation by overridingthese activity, the authors generated mutant flies that have either GroAA-- a stronger repressor, or GroDD -- a weak repressor when compared towildtype, and examined the functional relevance of the crosstalk betweenthese two pathways at the level/junction of Gro. The study showed thatwith a stronger repressor, there is a lack of vein formation in thedrosophila wing disc while flies with the weak repressor have enrichedvein formation. These findings clearly demonstrate the importance ofcrosstalk between two pathways in ensuring that the outcomes of downstream signaling leads to normal development and that a change in this junctionwill lead to dire functional consequences.

Student #7 Response to Student #6

May 19 2005 10:51AM

Student7 Student7

Cross-talk between the EGF receptor and Notch signaling pathways hasbeen documented previously, although the mechanism is poorly understood.Gro is a corepressor that acts with a variety of repressors, includingeffectors of the Notch, Wnt, and TGF-B pathways. It had been shownpreviously that there are genetic interactions between Gro and EGFRpathway, so the authors examine cross-talk between the EGFR and Notchpathways via Gro activity. They see an increase in Gro phosphorylationwith a constitutively active EGFR, or constitutively active MAPK. Theyalso showed that Gro was directly phosphorylated by MAPK in a kinaseassay. This phosphorylation of Gro decreases its ability as a repressor,and suggests that RTK signaling may downregulate Gro repressor activity.It had been shown that there is cross-talk between EGFR and Notch pathways during vein formation in wing patterning. The authors see that expressionof Gro that is nonphosphorylated results in a loss of veins in the wing,while expression of constitutively pseudo-phosphorylated Gro resulted inectopic vein material. This suggested that cross-talk between EGFR andNotch can occur at the level of Gro in this process. They also see thatphosphorylation of Gro plays a role in bristle formation, and suggest that regulation of Gro by EGFR is a basis for antagonism of Notch signaling.The data suggest that Gro is a point of intersection between the EGFR andNotch pathways. Downregulation of Gro by RTK phosphorylation would allowthe RTK pathway to alter transcriptional activity, so this paper shows how modification of a corepressor can link signaling and transcriptionalactivity. I agree with student's assessment that the paper doesdemonstrate cross-talk between two signaling pathways, and alsodemonstrates the functional consequences of this cross-talk.

Student #1 Response to Student #6

May 20 2005 10:18AM

student number 1

A global corepressor, Groucho (Gro) and its mammalian homologuesmediates many repression activities observed in other transcriptionrepressors. Gro and Gro-dependent repressors are downstream effectors ofseveral pathways (Paroush,Z. et al Cell, 1994; Giagtzoglou, N et alDevelopment, 2003; Chen, G. et al Gene, 2000). Thus, it is an interesting molecule to examine and most probable in its function as a signalintegration node between these different signal cascades. This paperelucidated the cross-talk between EGFR and Notch signaling pathways,observed previously (de Celis et al, Development, 1997; Zur Lag et al,Development, 1999), is mechanistically mediated via Gro. The authorsconvincingly demonstrated that RTK (ie. EGFR, MAPK) mediated Grophosphorylation resulted in decreased Gro repressive activity. Usingconstitutively active EGFR and kinase assay with synthetic phosphopeptideof the presumed phosphorylation site, the authors showed that Gro isindeed phosphorylated downstream of EGFR and specifically by Erk2 of MAPKpathway. Perhaps, the most convincing and biologically relevant data came from studies of Gro phosphorylation in Drosophila model system. Authorsfound that phospho-mimic Gro leads to additional wing vein development;while, the phosphor-deficient-mimic Gro leads to decreased wing veindevelopment. Analogous observation is made when authors examined notalbristles formations. These data, both in vitro and in vivo, are verystrong evidence that RTK pathway modulate Gro function via phosphorylation to antagonize Notch signaling. Thus, Groucho is the cross-talk nodebetween these two pathways.

I agree with other students that this paper demonstratedconvincingly, both via biochemical and animal studies, that Gro is thecrosstalk molecule between the EGFR and Notch signaling. Although, Iwould like to see what happens if one would knockout Groucho via tissuespecific siRNA constructs. I wonder if Groucho is the only molecule thatlinks EGFR and Notch signaling. Additionally, being a global corepressor, Groucho's phosphorylation status may have many unforeseen functions. Thus, I wonder if Gro's phosphorylation status may have an effect onother upstream pathways that feeds into Gro.


Chen, G. & Courey, A.J. Groucho/TLE family proteins and transcriptional repression. Gene 249, 1-16 2000. [PubMed] [Online Journal]

Cell. 1994 Dec 2;79(5):805-15. Groucho is required for Drosophila neurogenesis, segmentation, and sex determination and interacts directly with hairy-related bHLH proteins. Paroush Z, Finley RL Jr, Kidd T, Wainwright SM, Ingham PW, Brent R, Ish-Horowicz D. [PubMed]

Development. 2003 Jan;130(2):259-70. Two modes of recruitment of E(spl) repressors onto target genes. Giagtzoglou N, Alifragis P, Koumbanakis KA, Delidakis C.[PubMed] [Online Journal] [Full Text in Virtual Journal]

de Celis, J.F. & Bray, S. Garcia-Bellido, A. Notch signalling regulates veinlet expression and establishes boundaries between veins and interveins in the Drosophila wing. Development 124, 1919−1928 (1997). [PubMed] [Online Journal]

zur Lage, P. & Jarman, A.P. Antagonism of EGFR and notch signalling in the reiterative recruitment of Drosophila adult chordotonal sense organ precursors. Development 126, 3149−3157 (1999). [PubMed] [Online Journal]

Student #14 Response

May 20 2005 12:56PM

14 14

In this paper, the authors elucidate the previously unknown nature of the crosstalk between the EGFR and Notch pathways in Drosophila. In their model, the Gro protein sits at the junction of the EGFR and Notch. Theydemonstrate that increased phosphorylation of Gro by MAPK causes adecrease in Gro-mediated repression of Notch cascade proteins, andtherefore an increase in Notch-mediated transcription. The phenotypiceffect is that expression of a Gro mutant which cannot be phosphorylatedby MAPK causes a decrease in wing veins, while the expression of aconstitutively phosphorylated Gro mutant causes an increase in wing veins. Similar observations were noted in bristle development. The combinationof in-vitro and in-vivo data makes a strong case that the Gro proteinsserves as the junction between the EGFR and Notch pathways.

I agree with Student 4's assesement that this paper gives strongbiochemical, functional and phenotypic evidence of the crosstalk betweenthe Notch and EGFR pathways via the Gro protein. The paper is well- written and convicing, although to a non-Drosophilist audience it may have been helpful to have some additional explanation of the physiologicalreason for why EGFR would need to silence the Notch pathway. My onlyother concern is that the authors' biochemical examination of MAPKactivation is not entirely complete. In Figure 1, the authors used theDrosophila SL2 cell line for their experiments, which has increased levels of Ras and Raf, components of the MAPK pathway. (1) Consistenly withthis, in Figure 1c and 1f there is some phosphorylation of Gro inunstimulated cells. The authors claim that this is likely due to baseline MAPK activity, but this notion would be better supported if they showedthe increased phosphorylation of Ras following transfection, along withbaseline levels of Ras activity as a negative control.


Kimchie Z, Segev O, Lev Z. Maternal and embryonic transcripts of Drosophila proto-oncogenes areexpressed in Schneider 2 culture cells but not in l(2)gl transformedneuroblasts. Cell Differ Dev. 1989 Mar;26 [PubMed]

Final Forum: Student #1

May 10 2005 1:59PM

student number 01

1. The C2 domain of PKCdelta is a Phosphotyrosine Binding Domain, Cell,Vol. 121, 271-280 April 22, 2005.[PubMed] [Online Journal]
2. Phosphorylation and Functional inactivation of TSC2 by ERK:Implications for Tuberous Sclerosis and Cancer Pathogenesis, Cell, Vol.121, 179-193 April 22, 2005. [PubMed] [Online Journal]

Paper 1: Cellular signal transduction depends are protein-protein interactions that are regulated by post-translational modifications like phosphorylation,sumolation, acetylation, ubiquitination to name a few. Phosphorylation in particular is very well studied in the last decade, especially proteindomains that recognize phosphorylated residues. There appears to be twodistinct kinase groups that are responsible for cellular phosphorylationreactions, Serine/Theronine kinases and Tyrosine kinases. And associatedwith the respective phosphorylated residues are their phosphorylationbinding domains. For example, SH2 (src homology 2 domain) and PTB(phosphotyrosine binding domain) are well know for their affinity tophosphotyrosine residues; whereas, FHA (forkhead associated domains) and14-3-3 proteins binds to phosphoserine and phosphotheronine. However,until this paper, there is no evidence that phosphoserine/threoninekinases can bind to phosphotyrosine peptides or vice versa. This paper provides the first evidence that such interaction exist innature. PKCdelta, a Ser/Thr kinase, contains a C2 domain that binds tophosphotyrosine peptides. Previous research have shown thatPKCdelta interacts with Src (a tyrosine kinase), which led the authorsto surmise that there may be an intermediate protein that mediate thisinteraction. Indeed, they found that CDCP1, a transmembrane protein, is a Src substrate and mediates the interaction between Src and PKCdelta. Thescheme proposed is that Src phosphorylates CDCP1 on a tyrosine residue,which is the docking site for PKCdelta binding via its C2domain. This is confirmed via biochemical and structural studiesthat include x-ray crystallography of the association between C2 domainand phosphotyrosine peptides. This paper reveals a new mode ofinteraction between signal molecules that potentially have verysignificant consequences on cellular signaling networks. One can surmisethat there maybe a phosphotyrosin kinase that can bind tophosphorserine/theronine residues waiting to be characterized.

Paper 2: Tuberous sclerosis (TSC), an autosomal dominant tumor syndrome, is aresult of mutations in TSC1 and TSC2 genes. It has been establishedearlier that AKT is upstream of TSC1-2 complex and phosphorylate TSC1-2complex to inactive form. TSC1 and 2 heterodimeric complex suppressesmammalian target of rapamycin (MTOR). Previous research has shown thatTSC2 is a Rheb (ras homolog enriched in brain)-GAP where it activates Rheb -GTP hydolysis to Rheb-GDP. Rheb in its GTP-bound form activates mTOR,which leads to activation of p70s6k and eIF4E resulting in increasedtranslation. Thus, a distinct PI3K/AKT path of regulation is wellcharacterized. This paper demonstrates that in addition to this PI3K/AKTregulatory pathway, TSC2 is further regulated by MAPK pathway via Erk1/2. MAPK and PI3K/AKT pathways are two major pathways that are disrupted inhuman cancer. Finding a common regulated molecule like TSC2 in theirrespective cascade explains the clinical observation that in TSC2 normaltumors there is a correlative increase in Erk1/2 activity. Theexperiments in this paper have demonstrated that TSC2 is phosphorylated by Erk2 on S664 residue due to MAPK pathway activation. This phosphorylation leads to TSC1-2 complex dissociation and reduced activity of TSC2 towardsrepressing proliferation. The mouse studies conclusively demonstratedthat Erk phosphorylation on S664 leads to reduced TSC2 tumor suppressiveactivity. Thus, this signaling convergence leads to a biological andclinical relevant outcome.

Final Forum: Student #14

May 10 2005 2:02PM

014 014

Downregulation of lipopolysaccharide response in Drosophila bynegative crosstalk between the AP1 and NF-kappaB signaling modules.

Kim T, Yoon J, Cho H, Lee WB, Kim J, Song YH, Kim SN, Yoon JH, Kim-Ha J, Kim YJ. Nature Immunol. 2005 Feb;6(2):211-8. [PubMed] [Online Journal]

Drosophila has two pathways of innate immunity with which itrecognizes bacterial pathogens: the Toll pathway, which is triggered byLPS, and the Imd pathway, which is triggered by bacterial peptidoglycan(PGN) and is the focus of this paper. Imd activates dTAK, which thensignals to two different transcription factors. The first one is Relish(a Drosophila homolog of NFkB,) which is activated by the phosphorylationof IKK. The second is AP-1, which is activated via Basket (the Drosophila homolog of Jnk.) It has already been shown that NFkB activation candownregulate AP-1 activation under certain conditions. In this paper, theauthors show that the opposite is also true: AP-1 can downregulate NFkB- activated gene transcription. They demonstrate that the transcription ofthe Relish-dependent gene Attacin A is downregulated by AP-1. Themechanism involves the AP-1 transcription factor interaction with thehistone deacetylase HDAC1, and recruiting it to the gene’s Relishpromoter. The experiments, which involve microarray analysis, siRNA andChIPs, are well thought out and progress logically, although the figuresare not always clearly labeled.

Crosstalk between pathways can serve several different functions,often acting as a molecular switch between two different cell states or acoincidence detector. In this case, crosstalk acts a negative modulator,with two related pathways inhibiting each other’s transcription factorsafter several hours of activity. Since uncontrolled pro-inflammatorysignals in response to bacterial infection can lead to septic shock orautoimmune disease, it is crucial to have machinery for shutting off these signaling pathways. This paper is especially interesting becausecrosstalk usually involves protein modification such a ubiquitination orphosphorylation, whereas here it is accomplished by acetylation ofhistones and chromatin remodeling. This novel mechanism provides anelegant method of shutting off pro-inflammatory signaling while providinga sufficient time delay.

Student #4 response to Student #14

May 20 2005 7:53AM

Student 4 Student 4

The immune deficiency (Imd) pathway in Drosophila is responsiblefor immune responses to Gram negative bacteria. This paperdemonstrates cross-talk between the two branches of the Imdpathway, the Jnk (Basket) and NFkB (Relish) pathways. The NFkBpathway had previously been shown to negatively downregulatethe Jnk pathway in Drosophila (Park et al., 2004) as well as inmammals (Tang et al., 2001; De Smaele et al., 2001), butbidirectional cross-talk between these pathways had not beendemonstrated, although it had been suspected.

Using microarrays, the authors found that by knockdown of Basket(using RNAi) upregulated Relish-dependent genes, and vice- versa. This was confirmed using real-time PCR. They found thatactivator protein 1 (AP1), a chief target of Basket activation, wasresponsible for the respressive activity of Basket for Relish- dependent genes. Using ChIP, they showed that AP1 is recruitedto the promoter of a Relish-dependent gene, Attacin-A, and that itrecruits the histone deacetylase HDAC1 to downregulate Relish- activated Attacin-A transcription. Several other Relish-dependentgenes contain AP1 binding sites (and do not require Ap1 foractivation), suggesting that AP1 may act as a transcriptionalrepressor on a number of promoters. Overall, this paperconvincingly illustrates a mechanism for the Jnk pathway todownregulate the NFkB pathway by repressing the transcription ofgenes specific to that pathway. This cross-talk is thought toterminate signaling after an appropriate immune response, inorder to protect cells from an excess of harmful immunemodulating agents.

As the student states, the experiments in this paper are wellthought out and logical. The authors first look at global changes ingene expression using microarrays, then confirm these results anddissect out the precise mechanisms of cross-talk using othertechniques. I also agree that this paper demonstrates an unusualmethod of cross-talk. I think it is especially interesting that thecross-talk occurs at such a downstream point of the NFkB pathway,so that it ensures that the downregulation is highly selective to thatpathway. On the whole, I agree that this paper does effectivelyillustrate the concept of functional cross-talk between signalingpathways.


Park JM et al. 2004. Targeting of TAK1 by the NF-kB protein Relishregulates the JNK-mediated immune response in Drosophila. Genes Dev.18: 584-594 [PubMed] [Full Text in Virtual Journal] [Online Journal]

Tang G et al. 2001. Inhibition of JNK activation through NF-kB targetgenes. Nature 414: 313-317 [PubMed] [Online Journal]

De Smaele E et al. 2001. Induction of gadd45b by NF-kB downregulatespro-apoptotic JNK signaling. Nature 414: 308-313 [PubMed] [Online Journal]

Student #6 Response to Student #14

May 20 2005 10:31AM

student #6

IkB kinase (IKK) and Jun N-terminal kinase (Jnk) signaling modulesare important for the synthesis of immune effector molecules in innateimmune responses against lipopolysaccharide and peptidoglycan. It has been shown that NF-kB can negatively regulate Jnk pathways in both mammalianand drosophilia models[1-3]. However, it is not known if similar crosstalk occurs in the opposite direction: Can Jnk negatively regulates theactivities of NF-kB signaling modules? In this article, the authors,using a combination of microarray analysis, siRNA and ChIPs assay,demonstrated that crosstalk between drosophilia Jnk and IKK pathways atthe junction of AP1, involving histone aceytlation and chromatinremodeling, led to a downregulation of their activities. This articleprovided evidence of a bidirectional crosstalk and a novel mechanism –involvement of histone aceytlation and chromatin remodeling, that resultsin a negative regulation of the pathways involved.

I agree with the comments of Student #14 in that this paper isparticularly interesting as the crosstalk involves histone acetylation and chromatin remodeling, and the interaction between the two pathways is ofgreat importance for normal physiological responses.

1. De Smaele, E., et al., Induction of gadd45beta by NF-kappaBdownregulates pro-apoptotic JNK signalling. Nature, 2001. 414(6861): p.308-13. [PubMed] [Online Journal]

2. Park, J.M., et al., Targeting of TAK1 by the NF-kappa B protein Relishregulates the JNK-mediated immune response in Drosophila. Genes Dev, 2004. 18(5): p. 584-94.[PubMed] [Full Text in Virtual Journal] [Online Journal]

3. Tang, G., et al., Inhibition of JNK activation through NF-kappaB target genes. Nature, 2001. 414(6861): p. 313-7. [PubMed] [Online Journal]

Final Forum: Student #7

May 10 2005 5:19PM

Student7 Student7

Mori, S., Matsuzaki, K., Yoshida, K., Furukawa, F., Tahashi, Y.,Yamagata, H., Sekimoto, G., Seki, T., Matsui, H., Nishizawa, M., Fujisawa, J., and Okazaki, K. 2004. TGF-beta and HGF transmit signals through JNK- dependent Smad2/3 phosphorylation at the linker regions. Oncongene 23;7416-7429. [PubMed] [Online Journal]

The various signaling pathways that can become activated in cellshave very different consequences. Complicating this, many signalingpathways can interact with each other, and this also can lead to verydifferent outcomes for the cells. The paper by Mori, et al, demonstratescross-talk between two signaling pathways, and the functional consequences this can have on the cells. This paper deals with signaling cross-talkbetween TGF-b and HGF signaling pathways. TGF-b acts by binding to andactivating specific transmembrane receptor serine/threonine kinases, suchas TbR1. TbR1 then phosphorylates and activates R-Smads, which then couple to Smad4 and are translocated to the nucleus. In the nucleus, they canregulate transcription of specific genes. R-Smads have 2 conserveddomains, which are connected by a middle linker region. R-Smads have twomajor phosphorylation sites: in the C-terminal domain and in the linkerregion, and there is evidence to suggest that phosphorylation of Smads isimportant for their function. EGF and HGF, on the other hand, signalthrough transmembrane receptor tyrosine kinases. The most prominentpathways involve signaling through MAPKs, including ERK, JNK/SAPK1, andp38, which in turn activate transcription factors. Recent data hasreported Smad phosphorylation at linker regions upon HGF or EGF treatment, indicating that Smads play a role in mediating cross-talk between receptor serine kinases and receptor tyrosine kinases. In this paper, usingspecific phospho-antibodies to different Smad regions (C-terminal regionvs. linker regions), the authors showed that HGF and TGF-b both activatedthe JNK pathway, inducing endogenous phosphorylation at the linker regions of Smad 2 and 3. This suggests the linker regions are commonphosphorylation sites for HGF and TGF-b signaling pathways. Smad3phosphorylated by HGF increased its association with Smad4 and itstranslocation to the nucleus, which shows a biological function for linker region phosphorylation. This translocation can be blocked with aninhibitor of JNK. Treating the cells with HGF and TGF-b, the authors sawan increase in cell invasiveness, which was blocked with a JNK inhibitor.Because JNK can phosphorylate Smad3, it suggests that linker regionphosphorylation participates in the invasive ability of the cells.Although data has suggested that cross-talk between MAPK-mediated signalsand Smads occurs in the nucleus, the data in this paper suggests thatcross-talk can occur at the cytoplasmic level, with JNK directlyphosphorylating R-Smads, leading to transcriptional activation of the PAI- 1 gene in the nucleus. HGF can also inhibit the anti-proliferative effect of TGF-b, possibly due to decreased expression of p15INK4B, which may bedue to decreasing C-terminal phosphorylation of Smad3 (HGF decreases C- terminal phosphorylation of Smad3). Translocation of Smad3 to the nucleusupon HGF treatment could prevent C-terminal phosphorylation. This papershows that HGF cross-talk with the TGF-b pathway changes thephosphorylation state and cellular localization of R-Smads and, as aresult, cells may no longer respond in the same manner to TGF-b. Thiscould function, for example, in causing TGF-b to act as a tumor promoterinstead of a tumor suppressor. More work must be done to elucidate themolecular mechanisms of this signaling cross-talk, but this study isimportant because it shows that signaling pathway cross-talk can haveimportant functional consequences for the cell.

Response by Student #1

May 20 2005 10:27AM

student number 1

HGF (RTK signaling molecule) and TGFbeta (Receptor S/T Kinasesignaling molecule) are two important signaling molecules involved in cell invasion. Using primarily molecular techniques, Mori et al. studiedexhaustively the crosstalk between HGF and TGF-b pathways via Smadsignaling pathway. The generation of specific phospho-antibodies to theLinker and C-terminal regions of Smad2/3 is essential to this study. Theauthors convincingly demonstrated that HGF and TGF-b can activate the JNKpathways leading to phosphorylation of the Linker regions of Smad 2 and 3, which resulted in transactivation of PAI-1 gene leading to increasedinvasiveness of the cell. Additionally, HGF antagonize TGF-b anti- proliferative effects via inhibiting p15INK4B transcription, possibly dueto decreased Smad3 C-terminal phosphorylation. These findings aresupported by a series of inhibitor studies and luciferase assays.

I agree with the other students that this paper showed crosstalkbetween two very important pathways involved in tumor invasion process. Though, there is a lack of animal studies. I would like to see that thecrosstalk described by the authors can be replicated in the mouse model by observing the increased invasiveness of transplanted tumor cells. However, this does not detract from the paper’s significance ofdemonstrating the crosstalk between RTK and receptor ser/thr kinasepathways.

Student #4 response to Student #7

May 20 2005 10:27AM

Student 4 Student 4

This paper describes the functional cross-talk between the TGF- beta receptor (a receptor serine/threonine kinase) and thehepatocyte growth factor (HGF) receptor (a receptor tyrosinekinase). Receptor-activated Smads (R-Smads) are phosphorylatedby TGF-beta receptors and relay the receptor signal to the nucleus.Previous studies had implicated R-Smads in mediating the cross- talk between receptor serine kinases and receptor tyrosinekinases (Kretzschmar et al., 1997, 1999), but the precise molecularmechanisms and the functional consequences of this cross-talkwere not clear.

The authors of this paper used phospho-specific antibodies to R- Smads to demonstrate cross-talk between TGF-beta/Smad andHGF/MAPK/JNK-mediated signals. They determined that eitherHGF or TGF-beta treatment could phosphorylate R-Smads, andtogether produced an additive effect. The additive effect was foundto be due to additionally activated JNK, which directlyphosphorylated R-Smads at linker regions. Simultaneoustreatment with HGF and TGF-beta stimulated nuclear translocationof phosphorylated R-Smads, and increased the transcription ofplasminogen activator inhibitor type 1 (PAI-1), which is involved inTGF-beta-induced cell invasion. The in vivo relevance of this effectwas determined by measuring the invasive capacity induced byHGF and TGF-beta treatment, which was blocked by a JNKinhibitor. Thus, the authors were able to convincingly demonstratethe cross-talk between HGF and TGF-beta signaling pathways,producing a biologically relevant outcome.

I agree with the student’s choice of this paper as a good exampleof functional cross-talk between receptor serine/threonine kinaseand receptor tyrosine kinase pathways. The use of phospho- specific antibodies as a tool to reveal specific molecular alterationsand their consequences is particularly impressive. It is alsointeresting that, in this type of cross-talk, the two signalingpathways converge at a downstream cytoplasmic mediator, JNK,to produce an additive effect on PAI-1 transcription in the nucleus.This effect clearly has important implications in R-Smads-mediatedsignaling, and provides some insight into mechanisms of tumorinvasion.


Kretzschmar M, Doody J, Massague J. 1997. Opposing BMP andEGF signalling pathways converge on the TGF-beta familymediator Smad1. Nature 389: 618-622 [PubMed] [Online Journal]

Kretzschmar M, Doody J, Massague J. 1999. A mechanism ofrepression of TGFbeta/ Smad signaling by oncogenic Ras. GenesDev. 13: 804-816 [PubMed] [Full Text in Virtual Journal] [Online Journal]