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Degradation of FOS and JUN family proteins by calpains
Colloque SBCF-ATIPWCNRS DEGRADATION
OF FOS AND
JUN
FAMILY
Biologie du Dtveloppement, mars 1995
PROTEINS
GENETIC
ANALYSIS
OF
107
A TUMOR
SUPPRESSOR
GENE
LETHAL(2XIANTLARVAE fU2)GL). P.Manfruellit, W. Hanrattyz, M. Semerivat. 1 Laboratoire de GCn&ique et Physiologie du DCveloppement, Marseille-Luminy, Case Magali PARIAT, Serge CARILLO, Ann-Muriel STEFF, 907, 13288 Marseille cedex 9. France. Inactivation of the tumor suppressor gene [ethal(2)giant larvae Isabelle JARIEL-ENCONTRE, Pierre ROUX and Marc (l(2)gl) leads to an unrestricted pattern of growth in two specific cell PIECHACZYK types. Homozygous l(2)gl mutations cause the development of IGMM/UMR 9942. CNRS, BP 5051, Route de Mende, 34033, malignant tumors in the neuroblasts of the presumptive adult optic Montpellier Cedex 01, France center in the larval brain and in the imaeinal disc cells (Gateff. 1978). The l(2)& protein is a membrane-bou:d protein conckntrateh in tGe cell junctions and appears to be a phosphoprotein located at the c-fos is a transcription factor defining a multigene family in- cytoplasmic side of the membrane (Mechler and Strand, personal cluding fos-B, FRA- 1 and FRA-2, that has originally been found in two communication). retroviruses carrying mutated forms of the protein : FBT-MSV and In an attempt to dissect the signal transduction pathway in which l(2)gl is involved, we have undertaken a search for genes FBR-MSV. Fbs family members contribute to the AP-1 transcription whose products could interact in vivo with the l(2)gl protein. By using complex where they interact with members of the c-jun family : juntemperature-sensitive alleles of l(2)gl. we have initiated a mutagenesis B, c-jun and jun-II Cjun has originally been found in an avian onco- to identify second site mutations which would behave at restrictive genicretrovirus,ASV17, which carries a mutated form of the protein. temperature as dominant phenotypic suppressors. From about 20,000 Bgradation of c-fos protein (c-m) in the cytoplasm is rapid in EMS mutagenized chromosomes, we have recovered 3 mutations which suppress the 1(2)glts3/Dfnet 62 larval lethality. viva and contributes indirectly to the regula’tion of the protein Temperature-sensitive I(2)gl alleles have also been used to abondancy in the nucleus. We report that cytoplasmic c-m and c- reinvestigate the critical period for l(2)gl expression and we have JUN degradation can be initiated in a calcium-dependent manner shown that 1) the expression of 112Jal is reauired during the involving cysteine proteases called calpains. Interestingly, Fbs B, as embryogenesis; the embjonic developmk;t being ilocked duritg the well as all members of the jun family are also sensitive to calpains germ band retraction; 2) the maternal expression of l(2)gl is sufficient to allow a normal embrvogenesis to oroceed. In embrvos in which the albeit to different degrees. FRA-2 is resistant to micro-&pain maternal expression waslsuppresseb, a zygotic do& of l(2)gl was whereas FRA-I is resistant to both proteases. This thus adds an sufficient to obtain a normal development of the embryos; 3) a larval additional level to the regulation of the AP- 1 complex operating at the expression of l(2)gl seems to be necessary to produce viable adults; 4) finally, the l(2)gl function is required during oogenesis. cytoplasm level. The phenotypic analisys of the mutant embryos suggests that Pmmotifs are peptide regions commonly assumed to consti- the l(2)gl protein is a componant of the cytosqueleton and is involved tute signals for rapid recognition by calpains. Using site-directed mu- in the control of the cell shape and the acquisition of the polarity of the epithelial cells. tagenesis, we demonstrate that the 3 PEST motifs of c-m are dispensable for rapid proteolysis by calpains. athe mutated mviral proteins,v-Kl%FBRis resistant to calpains. Along the same line, the V-JUN-ASV17 is also less sensitive to calpains. We speculate that resistance of v-KE and v-JUN proteins to calpains may contribute to the tumorigenic potential of both FBR-MSV and ASVI 7. BY
CALPAINS
COMPARATIVE ANALYSIS OF THE TISSUE DISTRIBUTION OF THE THREE FIBROBLAST GROWTH FACTEUR RECEPTOR mRNA DURING AMPHIBIEN MORPHOGENESIS.
FROMENTOUX ValCrie, LAUNAY Catherine, THERY Clotilde, SHI De-Li and BOUCAUT Jean-Claude. Lnboratoire de Biologie Molkulaire et Cehlaire du Diveloppement, URA 1135, Groupe de Biologie Expe’rimentale, Vniversite’ P. et M. Curie, 9 quai Saint-Bernard, 75005 Paris, FRANCE.
We have used in situ hybridization to survey the expression pattern of three fibroblast.growth factor receptor (FGFR) mRNAs (PFR-1, PFRS and PFR-4 which we previously identified as the amphibian homoloes of human FGFR- I. FGFR-3 and FGFR-4 resuectivelv) during &orphogenesis. Previous work suggests that thdse FG$fi mRNAs exhibit a distinct pattern of expression at early developmental stages. In the nresent studv we test the functional activitv of these rec;ptors and show thai both acidic FGF(aFGF) and basic FGF(bFGF), but not KGF. can lead to their activation, suggesting that
\
,
III
I
HI9, A TUMOUR SUPPRESSOR GENE: EXPRESSION IN VARIOUS MAMMAL CELLS AND IN BREAST PRIMARY TUMOURS, TRANSLATABILITY OF A PUTATIVE 26 kDa PROTEIN *CURGY Jean Jacat e& **JOUBEL Anita,**WERNERT Nicolas **RAES MarietBertAe, **BEGUE Agnts *DUGIMONT Thieny: **LAGROU Christian,**S’IEHELIN Dokinique et **COLL Jean * Biologie cellulaire et moltkulaire du dkveloppement, SN3, Universitk de Lille I, 59655 Villeneuve d’Ascq.Cedex France. ** Oncologic molhdaire, CNRS VRA 1160, Institut Pasteur, I Rue Calmette, 59019 Lille Cedex France.
We previously reported the isolation of the human HI9 gene and its expression in various invasive cancers (DOUC-RASY et al. ,(1993), Intern. J. Oncol., 2, 753-758). The H19 gene is distinguishable by the following remarkable properties: high fetal expression, neonatal repression in almost all tissues, genomic imprinting (maternal allele only expressed), reexpression in numerous and various turnours, non-translated mRNA. The obvious discrepancy between the established phenotype of H19, as a tumour suppressor gene, and its reinitiated expression within various tumours prompted us to look for HI9 transcripts by in situ molecular hybridization in different breast turnours. This study indicated that: 1) HZ9 was highly expressed within the stromal component, occasionnaly at the stroma-epithelial cells interface, 2) transcription levels were different from one tumour to another, 3) cells expressing HZ9 were gathered in clusters. We focalised our study on the translatability of HI9 mRNA. The almost entire gene sequence and a truncated one (a large S’UTR deletion) were cloned and gave cDNA named ES and AP, respectively. After transfection of these cDNA in COS 1 cells, it appeared that AP cDNA only was translatable and gave a putative 26 kDa protein, from the longest open reading frame (ORF 4). Conditions of synthesis of this protein were considered after various 5’ or 3’ deletions of the S’UTR, as after point mutation of ORF3. Furthermore, two RNA binding proteins (average of 27 kDa) exhibited a high affinity for the S’UTR. In conclusion: 1) the HI9 mRNA abundance in some primary tumours could yield the activity of a mutated allele (a tumour suppressor role), 2) the repression of the HI9 mRNA translation from ORF4 could take its origin in several non-mutually exclusive causes: length and eventually secondary structures of the S’UTR, presence of 3 upstream ORF of which the last one seems to play a key-role, binding on the SUTR of two affin proteins.
OF FOS AND
JUN
FAMILY
Biologie du Dtveloppement, mars 1995
PROTEINS
GENETIC
ANALYSIS
OF
107
A TUMOR
SUPPRESSOR
GENE
LETHAL(2XIANTLARVAE fU2)GL). P.Manfruellit, W. Hanrattyz, M. Semerivat. 1 Laboratoire de GCn&ique et Physiologie du DCveloppement, Marseille-Luminy, Case Magali PARIAT, Serge CARILLO, Ann-Muriel STEFF, 907, 13288 Marseille cedex 9. France. Inactivation of the tumor suppressor gene [ethal(2)giant larvae Isabelle JARIEL-ENCONTRE, Pierre ROUX and Marc (l(2)gl) leads to an unrestricted pattern of growth in two specific cell PIECHACZYK types. Homozygous l(2)gl mutations cause the development of IGMM/UMR 9942. CNRS, BP 5051, Route de Mende, 34033, malignant tumors in the neuroblasts of the presumptive adult optic Montpellier Cedex 01, France center in the larval brain and in the imaeinal disc cells (Gateff. 1978). The l(2)& protein is a membrane-bou:d protein conckntrateh in tGe cell junctions and appears to be a phosphoprotein located at the c-fos is a transcription factor defining a multigene family in- cytoplasmic side of the membrane (Mechler and Strand, personal cluding fos-B, FRA- 1 and FRA-2, that has originally been found in two communication). retroviruses carrying mutated forms of the protein : FBT-MSV and In an attempt to dissect the signal transduction pathway in which l(2)gl is involved, we have undertaken a search for genes FBR-MSV. Fbs family members contribute to the AP-1 transcription whose products could interact in vivo with the l(2)gl protein. By using complex where they interact with members of the c-jun family : juntemperature-sensitive alleles of l(2)gl. we have initiated a mutagenesis B, c-jun and jun-II Cjun has originally been found in an avian onco- to identify second site mutations which would behave at restrictive genicretrovirus,ASV17, which carries a mutated form of the protein. temperature as dominant phenotypic suppressors. From about 20,000 Bgradation of c-fos protein (c-m) in the cytoplasm is rapid in EMS mutagenized chromosomes, we have recovered 3 mutations which suppress the 1(2)glts3/Dfnet 62 larval lethality. viva and contributes indirectly to the regula’tion of the protein Temperature-sensitive I(2)gl alleles have also been used to abondancy in the nucleus. We report that cytoplasmic c-m and c- reinvestigate the critical period for l(2)gl expression and we have JUN degradation can be initiated in a calcium-dependent manner shown that 1) the expression of 112Jal is reauired during the involving cysteine proteases called calpains. Interestingly, Fbs B, as embryogenesis; the embjonic developmk;t being ilocked duritg the well as all members of the jun family are also sensitive to calpains germ band retraction; 2) the maternal expression of l(2)gl is sufficient to allow a normal embrvogenesis to oroceed. In embrvos in which the albeit to different degrees. FRA-2 is resistant to micro-&pain maternal expression waslsuppresseb, a zygotic do& of l(2)gl was whereas FRA-I is resistant to both proteases. This thus adds an sufficient to obtain a normal development of the embryos; 3) a larval additional level to the regulation of the AP- 1 complex operating at the expression of l(2)gl seems to be necessary to produce viable adults; 4) finally, the l(2)gl function is required during oogenesis. cytoplasm level. The phenotypic analisys of the mutant embryos suggests that Pmmotifs are peptide regions commonly assumed to consti- the l(2)gl protein is a componant of the cytosqueleton and is involved tute signals for rapid recognition by calpains. Using site-directed mu- in the control of the cell shape and the acquisition of the polarity of the epithelial cells. tagenesis, we demonstrate that the 3 PEST motifs of c-m are dispensable for rapid proteolysis by calpains. athe mutated mviral proteins,v-Kl%FBRis resistant to calpains. Along the same line, the V-JUN-ASV17 is also less sensitive to calpains. We speculate that resistance of v-KE and v-JUN proteins to calpains may contribute to the tumorigenic potential of both FBR-MSV and ASVI 7. BY
CALPAINS
COMPARATIVE ANALYSIS OF THE TISSUE DISTRIBUTION OF THE THREE FIBROBLAST GROWTH FACTEUR RECEPTOR mRNA DURING AMPHIBIEN MORPHOGENESIS.
FROMENTOUX ValCrie, LAUNAY Catherine, THERY Clotilde, SHI De-Li and BOUCAUT Jean-Claude. Lnboratoire de Biologie Molkulaire et Cehlaire du Diveloppement, URA 1135, Groupe de Biologie Expe’rimentale, Vniversite’ P. et M. Curie, 9 quai Saint-Bernard, 75005 Paris, FRANCE.
We have used in situ hybridization to survey the expression pattern of three fibroblast.growth factor receptor (FGFR) mRNAs (PFR-1, PFRS and PFR-4 which we previously identified as the amphibian homoloes of human FGFR- I. FGFR-3 and FGFR-4 resuectivelv) during &orphogenesis. Previous work suggests that thdse FG$fi mRNAs exhibit a distinct pattern of expression at early developmental stages. In the nresent studv we test the functional activitv of these rec;ptors and show thai both acidic FGF(aFGF) and basic FGF(bFGF), but not KGF. can lead to their activation, suggesting that
\
,
III
I
HI9, A TUMOUR SUPPRESSOR GENE: EXPRESSION IN VARIOUS MAMMAL CELLS AND IN BREAST PRIMARY TUMOURS, TRANSLATABILITY OF A PUTATIVE 26 kDa PROTEIN *CURGY Jean Jacat e& **JOUBEL Anita,**WERNERT Nicolas **RAES MarietBertAe, **BEGUE Agnts *DUGIMONT Thieny: **LAGROU Christian,**S’IEHELIN Dokinique et **COLL Jean * Biologie cellulaire et moltkulaire du dkveloppement, SN3, Universitk de Lille I, 59655 Villeneuve d’Ascq.Cedex France. ** Oncologic molhdaire, CNRS VRA 1160, Institut Pasteur, I Rue Calmette, 59019 Lille Cedex France.
We previously reported the isolation of the human HI9 gene and its expression in various invasive cancers (DOUC-RASY et al. ,(1993), Intern. J. Oncol., 2, 753-758). The H19 gene is distinguishable by the following remarkable properties: high fetal expression, neonatal repression in almost all tissues, genomic imprinting (maternal allele only expressed), reexpression in numerous and various turnours, non-translated mRNA. The obvious discrepancy between the established phenotype of H19, as a tumour suppressor gene, and its reinitiated expression within various tumours prompted us to look for HI9 transcripts by in situ molecular hybridization in different breast turnours. This study indicated that: 1) HZ9 was highly expressed within the stromal component, occasionnaly at the stroma-epithelial cells interface, 2) transcription levels were different from one tumour to another, 3) cells expressing HZ9 were gathered in clusters. We focalised our study on the translatability of HI9 mRNA. The almost entire gene sequence and a truncated one (a large S’UTR deletion) were cloned and gave cDNA named ES and AP, respectively. After transfection of these cDNA in COS 1 cells, it appeared that AP cDNA only was translatable and gave a putative 26 kDa protein, from the longest open reading frame (ORF 4). Conditions of synthesis of this protein were considered after various 5’ or 3’ deletions of the S’UTR, as after point mutation of ORF3. Furthermore, two RNA binding proteins (average of 27 kDa) exhibited a high affinity for the S’UTR. In conclusion: 1) the HI9 mRNA abundance in some primary tumours could yield the activity of a mutated allele (a tumour suppressor role), 2) the repression of the HI9 mRNA translation from ORF4 could take its origin in several non-mutually exclusive causes: length and eventually secondary structures of the S’UTR, presence of 3 upstream ORF of which the last one seems to play a key-role, binding on the SUTR of two affin proteins.