Robert Ballotti - Academia.edu (original) (raw)

Papers by Robert Ballotti

Molecular Biology of the Cell, Jul 1, 1993

Reproduction Nutrition Development, 1989

Biochemical Journal, Jun 15, 1989

Biochemical Journal, 1987

Journal of Steroid Biochemistry, 1987

Molecular and Cellular Biochemistry, 1992

Using the synthetic peptide substrate Kemptide and cytosolic extracts of mouse fibroblasts transf... more Using the synthetic peptide substrate Kemptide and cytosolic extracts of mouse fibroblasts transfected with a human insulin receptor cDNA construct, we have studied an insulin-sensitive serine kinase activity. This activity is rapidly stimulated by insulin (maximum within 5 min) and also by orthovanadate. During cell extract preparation, para-nitrophenylphosphate and phosphotyrosine are able to preserve the enzyme activity, while phosphothreonine and phosphoserine fail to do so. Using antiphosphotyrosine antibodies, specific immunoprecipitation of this insulin- and orthovanadate-sensitive serine kinase was obtained. We then analysed by gel filtration chromatography eluates containing tyrosine-phosphorylated proteins obtained from unstimulated, insulin- and vanadate-treated cells. We found that several activities, with molecular weights estimated to be 30 kDa and smaller, are stimulated by both, insulin and orthovanadate. As a whole, our data indicate that insulin and orthovanadate enhance the cytosolic content in at least 2 or 3 phosphotyrosine-containing serine kinase activities.

Experimental Cell Research, 1991

In the present report we further approach the mechanism by which insulin and phenylarsine oxide (... more In the present report we further approach the mechanism by which insulin and phenylarsine oxide (PAO), a trivalent arsenical compound, regulate glucose transport in mouse fibroblasts (NIH3T3). First, we show that PAO is a powerful stimulatory agent on glucose transport. Second, at least three series of observations indicate that this action of PAO is not mediated through the insulin receptor: (i) the same effect of PAO is observed in NIH3T3 and in transfected cells expressing 6 x 10(6) insulin receptors, while the effect of insulin is markedly increased in the transfected cells; (ii) PAO does not affect the tyrosine phosphorylation of the insulin receptor; (iii) the tyrosine kinase activity of the insulin receptor toward exogenous substrates is not increased by PAO. Since PAO appears to act on glucose transport by a different mechanism than insulin, we have compared the effect of PAO and insulin on tyrosine phosphorylation of cellular proteins. Using Western blot analysis we did not detect common substrates in PAO- and insulin-treated cells. However, we found in cell extracts from both PAO- and insulin-treated cells a 50-kDa protein that is immunoprecipitated by antiphosphotyrosine antibody. In addition, PAO activates a cytosolic tyrosine kinase capable of poly(Glu/Tyr) phosphorylation. As a whole, our data suggest that the 50-kDa protein found in cells incubated with PAO and insulin could be the convergence point of the insulin and PAO signaling pathways.

Biochemistry, 1991

We have approached the functioning of a MAP kinase, which is thought to be a "sw... more We have approached the functioning of a MAP kinase, which is thought to be a "switch kinase" in the phosphorylation cascade initiated from various receptor tyrosine kinases including the insulin receptor. To do so, antipeptide antibodies were raised against the C-terminal portion of ERK1 (extracellular signal-regulated kinase 1), a protein kinase belonging to the family of MAP kinases. With these antipeptide antibodies, we observed the following: (i) a 44-kDa protein can be specifically recognized both under native and denaturing conditions; (ii) a 44-kDa phosphoprotein can be revealed in 32P-labeled cells; its phosphorylation is stimulated by insulin, sodium orthovanadate, and okadaic acid; (iii) a MBP kinase activity can be precipitated, which phosphorylates MBP on threonine residues, and which is stimulated by insulin, sodium orthovanadate, okadaic acid, and fetal calf serum; (iv) this MBP kinase activity appears to be correlated with the in vivo induced phosphorylation of the 44-kDa protein. We next studied the in vitro phosphorylation of this 44-kDa/ERK1-immunoreactive protein. A time- and manganese-dependent phosphorylation was stimulated by the in vitro addition of sodium orthovanadate. Phosphoamino acid analysis of the in vitro phosphorylated 44-kDa protein revealed both threonine and tyrosine phosphorylation. Importantly, this in vitro phosphorylation of MAP kinase results in activation of phosphorylation of added MBP substrate. As a whole, our data indicate that the 44-kDa phosphoprotein identified by our antipeptide antibodies very likely corresponds to a MAP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Journal of Biological Chemistry, May 1, 2008

The Journal of Immunology

Various cell surface receptors are phosphorylated upon binding of their ligand, and this phosphor... more Various cell surface receptors are phosphorylated upon binding of their ligand, and this phosphorylation seems to be involved in the signal transduction or in the feedback regulation of this signal. The possibility of a phosphorylation of the human IFN-gamma receptor (hu-IFN-gamma-R) has been investigated with 32P-labeled whole Raji cells and receptor purification either by immunoprecipitation with an anti-hu-IFN-gamma-R polyclonal antiserum or by affinity chromatography. The hu-IFN-gamma-R was found to be phosphorylated at a basal level. Upon incubation of the cells with recombinant hu-IFN-gamma, a dose-dependent two-fold increase of this phosphorylation was observed. Phosphoamino acid analysis by TLC showed that the same amino acids, serine and threonine, are phosphorylated at a basal level and after incubation with hu-IFN-gamma. Protein kinase C and Ca2+/calmodulin-dependent kinase pathways have been reported in some cases to be involved in the signal transduction pathway of hu-I...

Running title: CTNS controls melanin synthesis 2 Abbreviation list:

Je tiens à remercier mon directeur de thèse, le Pr. Simon Saule de m'avoir proposé ce sujet de re... more Je tiens à remercier mon directeur de thèse, le Pr. Simon Saule de m'avoir proposé ce sujet de recherche et de m'avoir aidé tout au long de mes 4 années de thèse. Nos discussions sur la biologie ont été très importantes et très enrichissantes dans mon travail même s'il m'arrivait d'être perdue parfois. Merci aussi pour m'avoir appris de nombreux proverbes oubliés de beaucoup d'entre nous ! Je tiens également à remercier le Dr. Emmanuel Barillot pour m'avoir encadrée et permis de réaliser ce travail dans son unité. L'entourage U900 a été d'une importance capitale dans l'avancement de mon projet. Je souhaite adresser mes remerciements aux membres de mon jury pour avoir accepté de juger mon travail. Merci au Dr. Philippe Dessen et au Dr. Robert Ballotti d'avoir consacré du temps à rapporter mon manuscrit, au Pr. Jessica Zucman-Rossi et au Pr. Frédéric Mouriaux mes examinateurs ainsi qu'au Dr. Daniel Gautheret qui a accepté d'en être le président. Je remercie l'ensemble des membres du projet mélanome uvéal avec qui j'ai interagit tout au long de mon doctorat. Un grand merci à

Cell Death & Disease, 2021

In the search of biguanide-derived molecules against melanoma, we have discovered and developed a... more In the search of biguanide-derived molecules against melanoma, we have discovered and developed a series of bioactive products and identified the promising new compound CRO15. This molecule exerted anti-melanoma effects on cells lines and cells isolated from patients including the ones derived from tumors resistant to BRAF inhibitors. Moreover, CRO15 was able to decrease viability of cells lines from a broad range of cancer types. This compound acts by two distinct mechanisms. First by activating the AMPK pathway induced by a mitochondrial disorder. Second by inhibition of MELK kinase activity, which induces cell cycle arrest and activation of DNA damage repair pathways by p53 and REDD1 activation. All of these mechanisms activate autophagic and apoptotic processes resulting in melanoma cell death. The strong efficacy of CRO15 to reduce the growth of melanoma xenograft sensitive or resistant to BRAF inhibitors opens interesting perspective.

Journal of Biological Chemistry, 1990

The EMBO Journal, 1987

Communicated by M.Raff 1987). In adult rat and human brain the two types of receptor are present ... more Communicated by M.Raff 1987). In adult rat and human brain the two types of receptor are present (Gammeltoft et al., 1985). These observations raise questions about the cellular origin of IGFs in fetal and adult brain, the nature of their target cells in the CNS and their molecular mechanisms of action on brain cells, in particular the role of the type I IGF receptor tyrosine kinase. Glial cells constitute a major cellular component of the CNS, and regulation of glial cell growth is clearly important in brain development (Raff et al., 1983) and in repair of brain injury (Guilian et al., 1986). In this study we describe the expression of the IGF I gene in cultured astrocytes, and the interaction of IGF I with the same cells. Our data suggest that astroglial cells

Biochemical Journal, 1985

Addition of insulin to wheat-germ-lectin-purified glycoproteins derived from rat hepatocytes or r... more Addition of insulin to wheat-germ-lectin-purified glycoproteins derived from rat hepatocytes or rabbit brown adipose tissue results in the increased phosphorylation of a Mr-110 000 protein. This naturally occurring glycoprotein appears as a monomeric structure and is not part of the insulin receptor itself, since it is not immunoprecipitated by highly specific antibodies to insulin receptor. Phosphorylation of the Mr-110 000 protein and autophosphorylation of the receptor beta-subunit (Mr 95 000) are stimulated by insulin in a remarkably similar dose-dependent fasion, with half-maximal stimulation at 1 nM-insulin. Further, kinetic studies suggest that the phosphorylation of the Mr-110 000 protein occurs after autophosphorylation of the insulin-receptor kinase. In conclusion, the present identification of an endogenous substrate for the insulin-receptor kinase could suggest that some, if not all, effects of insulin may be mediated through activation of this kinase.

Journal of the American Academy of Dermatology, 2015

Background: It can be useful to assess the NRAS mutation status in patients with metastatic melan... more Background: It can be useful to assess the NRAS mutation status in patients with metastatic melanoma because NRAS-activating mutations confer resistance to RAF inhibitors, and NRAS-mutated patients appear to be sensitive to mitogen-activated protein kinase (MEK) inhibitors. Objective: We aimed to assess the diagnostic accuracy of an immunohistochemistry (IHC) approach using a novel anti-NRAS (Q61R) monoclonal antibody on formalin-fixed paraffin-embedded tissue samples from patients with metastatic melanoma. Methods: We conducted a retrospective multicenter cohort study on 170 patients with metastatic melanoma. The automated IHC assay was performed using the SP174 clone, and compared with results of the molecular testing. Results: Evaluation of a test cohort with knowledge of the mutation status established a specific IHC pattern for the mutation. In the independent blinded analysis of the remaining cases, the anti-NRAS (Q61R) antibody accurately identified all NRAS Q61R-mutated tumors, and demonstrated 100% sensitivity and specificity. Limitations: Limitations include retrospective design and lack of multicenter interobserver reproducibility. Conclusion: The NRAS (Q61R) IHC assay is reliable and specific for the evaluation of the Q61R mutation status in metastatic melanoma and may be an alternative to molecular biology in evaluation of metastatic melanoma in routine practice.

The Journal of cell biology, 1992

Cell surface tyrosine kinase receptors are subject to a rapid activation by their ligand, which i... more Cell surface tyrosine kinase receptors are subject to a rapid activation by their ligand, which is followed by secondary regulatory processes. The IHE2 cell line is a unique model system to study the regulation of EGF binding to EGF receptors after activation of the EGF receptor kinase. IHE2 cells express both a chimeric insulin-EGF receptor kinase (IER) and a kinase-deficient EGF receptor (HER K721A). We have previously reported that IER is an insulin-responsive EGF receptor tyrosine kinase that activates one or several serine/threonine kinases, which in turn phosphorylate(s) the unoccupied HER K721A. In this article we show that insulin through IER activation induces a decrease in 125I-EGF binding to IHE2 cells. Scatchard analysis indicates that, as for TPA, the effect of insulin can be accounted for by a loss of the high affinity binding of EGF to HER K721A. Since this receptor transmodulation persists in protein kinase C downregulated IHE2 cells, it is likely to be due to a mech...

Molecular Biology of the Cell, Jul 1, 1993

Reproduction Nutrition Development, 1989

Biochemical Journal, Jun 15, 1989

Biochemical Journal, 1987

Journal of Steroid Biochemistry, 1987

Molecular and Cellular Biochemistry, 1992

Using the synthetic peptide substrate Kemptide and cytosolic extracts of mouse fibroblasts transf... more Using the synthetic peptide substrate Kemptide and cytosolic extracts of mouse fibroblasts transfected with a human insulin receptor cDNA construct, we have studied an insulin-sensitive serine kinase activity. This activity is rapidly stimulated by insulin (maximum within 5 min) and also by orthovanadate. During cell extract preparation, para-nitrophenylphosphate and phosphotyrosine are able to preserve the enzyme activity, while phosphothreonine and phosphoserine fail to do so. Using antiphosphotyrosine antibodies, specific immunoprecipitation of this insulin- and orthovanadate-sensitive serine kinase was obtained. We then analysed by gel filtration chromatography eluates containing tyrosine-phosphorylated proteins obtained from unstimulated, insulin- and vanadate-treated cells. We found that several activities, with molecular weights estimated to be 30 kDa and smaller, are stimulated by both, insulin and orthovanadate. As a whole, our data indicate that insulin and orthovanadate enhance the cytosolic content in at least 2 or 3 phosphotyrosine-containing serine kinase activities.

Experimental Cell Research, 1991

In the present report we further approach the mechanism by which insulin and phenylarsine oxide (... more In the present report we further approach the mechanism by which insulin and phenylarsine oxide (PAO), a trivalent arsenical compound, regulate glucose transport in mouse fibroblasts (NIH3T3). First, we show that PAO is a powerful stimulatory agent on glucose transport. Second, at least three series of observations indicate that this action of PAO is not mediated through the insulin receptor: (i) the same effect of PAO is observed in NIH3T3 and in transfected cells expressing 6 x 10(6) insulin receptors, while the effect of insulin is markedly increased in the transfected cells; (ii) PAO does not affect the tyrosine phosphorylation of the insulin receptor; (iii) the tyrosine kinase activity of the insulin receptor toward exogenous substrates is not increased by PAO. Since PAO appears to act on glucose transport by a different mechanism than insulin, we have compared the effect of PAO and insulin on tyrosine phosphorylation of cellular proteins. Using Western blot analysis we did not detect common substrates in PAO- and insulin-treated cells. However, we found in cell extracts from both PAO- and insulin-treated cells a 50-kDa protein that is immunoprecipitated by antiphosphotyrosine antibody. In addition, PAO activates a cytosolic tyrosine kinase capable of poly(Glu/Tyr) phosphorylation. As a whole, our data suggest that the 50-kDa protein found in cells incubated with PAO and insulin could be the convergence point of the insulin and PAO signaling pathways.

Biochemistry, 1991

We have approached the functioning of a MAP kinase, which is thought to be a "sw... more We have approached the functioning of a MAP kinase, which is thought to be a "switch kinase" in the phosphorylation cascade initiated from various receptor tyrosine kinases including the insulin receptor. To do so, antipeptide antibodies were raised against the C-terminal portion of ERK1 (extracellular signal-regulated kinase 1), a protein kinase belonging to the family of MAP kinases. With these antipeptide antibodies, we observed the following: (i) a 44-kDa protein can be specifically recognized both under native and denaturing conditions; (ii) a 44-kDa phosphoprotein can be revealed in 32P-labeled cells; its phosphorylation is stimulated by insulin, sodium orthovanadate, and okadaic acid; (iii) a MBP kinase activity can be precipitated, which phosphorylates MBP on threonine residues, and which is stimulated by insulin, sodium orthovanadate, okadaic acid, and fetal calf serum; (iv) this MBP kinase activity appears to be correlated with the in vivo induced phosphorylation of the 44-kDa protein. We next studied the in vitro phosphorylation of this 44-kDa/ERK1-immunoreactive protein. A time- and manganese-dependent phosphorylation was stimulated by the in vitro addition of sodium orthovanadate. Phosphoamino acid analysis of the in vitro phosphorylated 44-kDa protein revealed both threonine and tyrosine phosphorylation. Importantly, this in vitro phosphorylation of MAP kinase results in activation of phosphorylation of added MBP substrate. As a whole, our data indicate that the 44-kDa phosphoprotein identified by our antipeptide antibodies very likely corresponds to a MAP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Journal of Biological Chemistry, May 1, 2008

The Journal of Immunology

Various cell surface receptors are phosphorylated upon binding of their ligand, and this phosphor... more Various cell surface receptors are phosphorylated upon binding of their ligand, and this phosphorylation seems to be involved in the signal transduction or in the feedback regulation of this signal. The possibility of a phosphorylation of the human IFN-gamma receptor (hu-IFN-gamma-R) has been investigated with 32P-labeled whole Raji cells and receptor purification either by immunoprecipitation with an anti-hu-IFN-gamma-R polyclonal antiserum or by affinity chromatography. The hu-IFN-gamma-R was found to be phosphorylated at a basal level. Upon incubation of the cells with recombinant hu-IFN-gamma, a dose-dependent two-fold increase of this phosphorylation was observed. Phosphoamino acid analysis by TLC showed that the same amino acids, serine and threonine, are phosphorylated at a basal level and after incubation with hu-IFN-gamma. Protein kinase C and Ca2+/calmodulin-dependent kinase pathways have been reported in some cases to be involved in the signal transduction pathway of hu-I...

Running title: CTNS controls melanin synthesis 2 Abbreviation list:

Je tiens à remercier mon directeur de thèse, le Pr. Simon Saule de m'avoir proposé ce sujet de re... more Je tiens à remercier mon directeur de thèse, le Pr. Simon Saule de m'avoir proposé ce sujet de recherche et de m'avoir aidé tout au long de mes 4 années de thèse. Nos discussions sur la biologie ont été très importantes et très enrichissantes dans mon travail même s'il m'arrivait d'être perdue parfois. Merci aussi pour m'avoir appris de nombreux proverbes oubliés de beaucoup d'entre nous ! Je tiens également à remercier le Dr. Emmanuel Barillot pour m'avoir encadrée et permis de réaliser ce travail dans son unité. L'entourage U900 a été d'une importance capitale dans l'avancement de mon projet. Je souhaite adresser mes remerciements aux membres de mon jury pour avoir accepté de juger mon travail. Merci au Dr. Philippe Dessen et au Dr. Robert Ballotti d'avoir consacré du temps à rapporter mon manuscrit, au Pr. Jessica Zucman-Rossi et au Pr. Frédéric Mouriaux mes examinateurs ainsi qu'au Dr. Daniel Gautheret qui a accepté d'en être le président. Je remercie l'ensemble des membres du projet mélanome uvéal avec qui j'ai interagit tout au long de mon doctorat. Un grand merci à

Cell Death & Disease, 2021

In the search of biguanide-derived molecules against melanoma, we have discovered and developed a... more In the search of biguanide-derived molecules against melanoma, we have discovered and developed a series of bioactive products and identified the promising new compound CRO15. This molecule exerted anti-melanoma effects on cells lines and cells isolated from patients including the ones derived from tumors resistant to BRAF inhibitors. Moreover, CRO15 was able to decrease viability of cells lines from a broad range of cancer types. This compound acts by two distinct mechanisms. First by activating the AMPK pathway induced by a mitochondrial disorder. Second by inhibition of MELK kinase activity, which induces cell cycle arrest and activation of DNA damage repair pathways by p53 and REDD1 activation. All of these mechanisms activate autophagic and apoptotic processes resulting in melanoma cell death. The strong efficacy of CRO15 to reduce the growth of melanoma xenograft sensitive or resistant to BRAF inhibitors opens interesting perspective.

Journal of Biological Chemistry, 1990

The EMBO Journal, 1987

Communicated by M.Raff 1987). In adult rat and human brain the two types of receptor are present ... more Communicated by M.Raff 1987). In adult rat and human brain the two types of receptor are present (Gammeltoft et al., 1985). These observations raise questions about the cellular origin of IGFs in fetal and adult brain, the nature of their target cells in the CNS and their molecular mechanisms of action on brain cells, in particular the role of the type I IGF receptor tyrosine kinase. Glial cells constitute a major cellular component of the CNS, and regulation of glial cell growth is clearly important in brain development (Raff et al., 1983) and in repair of brain injury (Guilian et al., 1986). In this study we describe the expression of the IGF I gene in cultured astrocytes, and the interaction of IGF I with the same cells. Our data suggest that astroglial cells

Biochemical Journal, 1985

Addition of insulin to wheat-germ-lectin-purified glycoproteins derived from rat hepatocytes or r... more Addition of insulin to wheat-germ-lectin-purified glycoproteins derived from rat hepatocytes or rabbit brown adipose tissue results in the increased phosphorylation of a Mr-110 000 protein. This naturally occurring glycoprotein appears as a monomeric structure and is not part of the insulin receptor itself, since it is not immunoprecipitated by highly specific antibodies to insulin receptor. Phosphorylation of the Mr-110 000 protein and autophosphorylation of the receptor beta-subunit (Mr 95 000) are stimulated by insulin in a remarkably similar dose-dependent fasion, with half-maximal stimulation at 1 nM-insulin. Further, kinetic studies suggest that the phosphorylation of the Mr-110 000 protein occurs after autophosphorylation of the insulin-receptor kinase. In conclusion, the present identification of an endogenous substrate for the insulin-receptor kinase could suggest that some, if not all, effects of insulin may be mediated through activation of this kinase.

Journal of the American Academy of Dermatology, 2015

Background: It can be useful to assess the NRAS mutation status in patients with metastatic melan... more Background: It can be useful to assess the NRAS mutation status in patients with metastatic melanoma because NRAS-activating mutations confer resistance to RAF inhibitors, and NRAS-mutated patients appear to be sensitive to mitogen-activated protein kinase (MEK) inhibitors. Objective: We aimed to assess the diagnostic accuracy of an immunohistochemistry (IHC) approach using a novel anti-NRAS (Q61R) monoclonal antibody on formalin-fixed paraffin-embedded tissue samples from patients with metastatic melanoma. Methods: We conducted a retrospective multicenter cohort study on 170 patients with metastatic melanoma. The automated IHC assay was performed using the SP174 clone, and compared with results of the molecular testing. Results: Evaluation of a test cohort with knowledge of the mutation status established a specific IHC pattern for the mutation. In the independent blinded analysis of the remaining cases, the anti-NRAS (Q61R) antibody accurately identified all NRAS Q61R-mutated tumors, and demonstrated 100% sensitivity and specificity. Limitations: Limitations include retrospective design and lack of multicenter interobserver reproducibility. Conclusion: The NRAS (Q61R) IHC assay is reliable and specific for the evaluation of the Q61R mutation status in metastatic melanoma and may be an alternative to molecular biology in evaluation of metastatic melanoma in routine practice.

The Journal of cell biology, 1992

Cell surface tyrosine kinase receptors are subject to a rapid activation by their ligand, which i... more Cell surface tyrosine kinase receptors are subject to a rapid activation by their ligand, which is followed by secondary regulatory processes. The IHE2 cell line is a unique model system to study the regulation of EGF binding to EGF receptors after activation of the EGF receptor kinase. IHE2 cells express both a chimeric insulin-EGF receptor kinase (IER) and a kinase-deficient EGF receptor (HER K721A). We have previously reported that IER is an insulin-responsive EGF receptor tyrosine kinase that activates one or several serine/threonine kinases, which in turn phosphorylate(s) the unoccupied HER K721A. In this article we show that insulin through IER activation induces a decrease in 125I-EGF binding to IHE2 cells. Scatchard analysis indicates that, as for TPA, the effect of insulin can be accounted for by a loss of the high affinity binding of EGF to HER K721A. Since this receptor transmodulation persists in protein kinase C downregulated IHE2 cells, it is likely to be due to a mech...