The intrinsic protein tyrosine kinase activity of the epidermal growth factor (EGF) receptor is n... more The intrinsic protein tyrosine kinase activity of the epidermal growth factor (EGF) receptor is necessary for ligand-induced signaling. To determine whether cellular protein tyrosine kinases are substrates for EGF-activated receptors, phosphotyrosine-containing proteins were isolated from EGF-treated cells and assayed for tyrosine kinase activity using peptide substrates. A tyrosine kinase activity that is distinct from the EGF receptor was adsorbed to monoclonal anti-phosphotyrosine antibody columns and eluted with phenyl phosphate. Near-maximal tyrosine phosphorylation of this kinase occurred within 1 min of cell stimulation with an ED50 for EGF of 2.5 nM. The kinase was deactivated by incubation with purified CD45 tyrosine phosphatase in vitro, but activity could be restored by incubation with purified EGF receptor and Mn2+ ATP. These results suggest a cascade of tyrosine kinase signaling analogous to well characterized serine/threonine kinase cascades.
We have developed a method to study the primary sequence specificities of protein kinases by usin... more We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.
Cells expressing mutant epidermal growth factor (EGF) receptors have been used to study mechanism... more Cells expressing mutant epidermal growth factor (EGF) receptors have been used to study mechanisms through which EGF increases phospholipase C (PLC) activity. C-terminal truncation mutant EGF receptors are markedly impaired in their ability to increase inositol phosphate formation compared with wild-type EGF receptors. Mutation of the single tyrosine self-phosphorylation site at residue 992 to phenylalanine in an EGF receptor truncated at residue 1000 abolished the ability of EGF to increase inositol phosphate formation. C-terminal deletion mutant receptors that are impaired in their ability to increase inositol phosphate formation effectively phosphorylate PLC-gamma at the same tyrosine residues as do wild-type EGF receptors. EGF enhances PLC-gamma association with wild-type EGF receptors but not with mutant receptors lacking sites of tyrosine phosphorylation. These results indicate that formation of a complex between self-phosphorylated EGF receptors and PLC-gamma is necessary for...
Considerable effort is currently being devoted to understand the functions of protein p53, a majo... more Considerable effort is currently being devoted to understand the functions of protein p53, a major regulator of cell proliferation. The protein p53 has been reported to catalyse the annealing of complementary DNA or RNA strands. We report that this activity is inhibited in the presence of the serine/threonine protein kinase CK2. It is shown that this inhibition can be explained by the occurrence of a high-affinity molecular association between p53 and CK2. The molecular complex involves an interaction between the C-terminal domain of p53 and the β subunit of the oligomeric kinase. Accordingly, the isolated α subunit of the kinase was without effect. In addition, after phosphorylation by CK2, phosphorylated p53 lost its DNA annealing activity. Because the C-terminal domain of p53 is both involved in the association with CK2 and phosphorylated by it, our results suggest that either protein–protein interaction or phosphorylation of this domain might control the base pairing of compleme...
The Fas associated death domain protein (FADD) is the key adaptor molecule of the apoptotic signa... more The Fas associated death domain protein (FADD) is the key adaptor molecule of the apoptotic signal triggered by death receptors of the TNF-R1 superfamily. Besides its crucial role in the apoptotic machinery, FADD has proved to be important in many biological processes like tumorigenesis, embryonic development or cell cycle progression. In a process to decipher the regulatory mechanisms underlying FADD regulation, we identified the anti-apoptotic kinase, CK2, as a new partner and regulator of FADD sub-cellular localization. The blockade of CK2 activity induced FADD re-localization within the cell. Moreover, cytoplasmic FADD was increased when CK2β was knocked down. In vitro kinase and pull down assays confirmed that FADD could be phosphorylated by the CK2 holoenzyme. We found that phosphorylation is weak with CK2α alone and optimal in the presence of stoichiometric amounts of CK2α catalytic and CK2β regulatory subunits, showing that FADD phosphorylation is undertaken by the CK2 holoe...
A Molecular and Cellular View of Protein Kinase CK2, 1999
To date, the intracellular regulation of protein kinase CK2 is unknown. However it was observed t... more To date, the intracellular regulation of protein kinase CK2 is unknown. However it was observed that the enzyme associates with several intracellular proteins and the formation of such molecular complexes may represent a mechanism for the control of CK2 activity. Using the Interaction Trap system in yeast, with the CK2β as a bait, we looked for CK2 partners. We present the identification of new potential partners of CK2β and it is hoped that their classification will help in understanding the physiological roles and the regulation of CK2 in the cell.
A Molecular and Cellular View of Protein Kinase CK2, 1999
We have characterized several subdomains of the β subunit of protein kinase CK2. The N-terminal h... more We have characterized several subdomains of the β subunit of protein kinase CK2. The N-terminal half of the protein exhibits a pseudo-substrate segment in tandem with a polyamine binding domain responsible for the activation of the kinase by these polybasic compounds. Study of the chemical features of this polyamine binding site showed that polyamine analogs exhibiting the highest affinity for CK2 are the best CK2 activators. Mutational analysis disclosed that glutamic residues lying in the polyacidic region of the CK2β subunit are involved in the interaction with polyamine molecules and allowed the delineation of an autonomous binding domain. Furthermore, this regulatory domain was shown to mediate the association of CK2 with plasma membrane. The C-terminal domain of the CK2β subunit plays a role in the oligomerization of the kinase since it was observed that a truncated form of this subunit lacking its 33-last amino acids was incompetent for the assembly of polymeric forms of CK2. Altogether, our results support the notion that the β subunit of CK2 is a modular protein made by the association of interdependent domains that are involved in its multiple functions.
Protein Kinase CK2 — From Structure to Regulation, 2001
We have generated fusion proteins between the subunits of CK2 and GFP and characterized their beh... more We have generated fusion proteins between the subunits of CK2 and GFP and characterized their behaviour in living cells. The expressed fusion proteins were functional and interacted with endogenous CK2. Imaging of NIH3T3 cells expressing low level of GFP-CK2α or GFP-CK2β showed that both proteins were mostly nuclear in interphase. Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Once in the nucleus, both subunits diffused rapidly in the nucleoplasm. In mitotic cells, CK2 subunits were dispersed throughout the cytoplasm and were not associated to chromatin. Our data are compatible with the idea that each subunit can translocate individually to the nucleus to interact with each other or with important cellular partners. Understanding the molecular mechanisms which regulate the dynamic localization of CK2 subunits will be of central importance.
Protein kinase CK2 is a tetramer composed of two α catalytic subunits and two β regulatory subuni... more Protein kinase CK2 is a tetramer composed of two α catalytic subunits and two β regulatory subunits. The structure of a C-terminal truncated form of the human β subunit has been determined by X-ray crystallography to 1.7 Å resolution. One dimer is observed in the asymmetric unit of the crystal. The most striking feature of the structure is the presence of a zinc finger mediating the dimerization. The monomer structure consists of two domains, one entirely α-helical and one including the zinc finger. The dimer has a crescent shape holding a highly acidic region at both ends. We propose that this acidic region is involved in the interactions with the polyamines and/or catalytic subunits. Interestingly, conserved amino acid residues among β subunit sequences are clustered along one linear ridge that wraps around the entire dimer. This feature suggests that protein partners may interact with the dimer through a stretch of residues in an extended conformation.
Protein kinase CK2 is a multi-subunit complex whose dynamic assembly appears as a crucial point o... more Protein kinase CK2 is a multi-subunit complex whose dynamic assembly appears as a crucial point of regulation. The ability to interfere with specific protein-protein interactions has already provided powerful means of influencing the functions of selected proteins within the cell. CK2beta-derived cyclopeptides that target a well-defined hydrophobic pocket on CK2alpha have been previously characterized as potent inhibitors of CK2 subunit assembly [9]. As a first step toward the rational design of low molecular weight CK2 antagonists, we have in the present study screened a collection of podophyllotoxine indolo-analogues to identify chemical inhibitors of the CK2 subunit interaction. We report the identification of a podophyllotoxine indolo-analogue as a chemical ligand that binds to the CK2alpha/CK2beta interface inducing selective disruption of the CK2alpha/CK2beta assembly and concomitant inhibition of CK2alpha activity.
The Epstein-Barr Virus (EBV) early protein EB2 (also called BMLF1, Mta, or SM) promotes the nucle... more The Epstein-Barr Virus (EBV) early protein EB2 (also called BMLF1, Mta, or SM) promotes the nuclear export of a subset of early and late viral mRNAs and is essential for the production of infectious virions. We show here that in vitro, protein kinase CK2α and -β subunits bind both individually and, more efficiently, as a complex to the EB2 N terminus and that the CK2β regulatory subunit also interacts with the EB2 C terminus. Immunoprecipitated EB2 has CK2 activity that phosphorylates several sites within the 80 N-terminal amino acids of EB2, including Ser-55, -56, and -57, which are localized next to the nuclear export signal. EB2S3E, the phosphorylation-mimicking mutant of EB2 at these three serines, but not the phosphorylation ablation mutant EB2S3A, efficiently rescued the production of infectious EBV particles by HEK293 BMLF1-KO cells harboring an EB2-defective EBV genome. The defect of EB2S3A in transcomplementing 293 BMLF1-KO cells was not due to impaired nucleocytoplasmic sh...
1 The abbreviations used are: CK2, protein kinase CK2; MBP, maltose-binding protein; MBP51-110, ... more 1 The abbreviations used are: CK2, protein kinase CK2; MBP, maltose-binding protein; MBP51-110, fusion protein of MBP and the CK2  subunit domain Asp 51-Pro 110 ; MBP1-215, fusion protein of MBP and the entire CK2  subunit; MBP3, fusion protein of MBP and the entire CK2 Ala60,Ala61,Ala63 subunit; HA, fusion of the hemagglutinin tag at the C terminus of the CK2 regulatory subunit; 3HA, fusion of the hemagglutinin tag at the C terminus of the Ala60,Ala61,Ala63 subunit; CD, circular dichroism; PCR, polymerase chain reaction; BSA, bovine serum albumin.
The presence of fibroblast growth factor-2 (FGF-2) in the nucleus has now been reported both in v... more The presence of fibroblast growth factor-2 (FGF-2) in the nucleus has now been reported both in vitro and in vivo, but its nuclear functions are unknown. Here, we show that FGF-2 added to nuclear extract binds to protein kinase CK2 and nucleolin, a CK2 natural substrate. Added to baculovirus-infected cell extracts overexpressing CK2 or its isolated subunits, FGF-2 binds to the enzyme through its regulatory  subunit. Using purified proteins, FGF-2 is shown to directly interact with CK2 and to stimulate CK2 activity toward nucleolin. Furthermore, a mitogenic-deficient FGF-2 mutant protein has an impaired ability to interact with CK2 and to stimulate CK2 activity using nucleolin as substrate. We propose that in growing cells, one function of nuclear FGF-2 is to modulate CK2 activity through binding to its regulatory  subunit.
The intrinsic protein tyrosine kinase activity of the epidermal growth factor (EGF) receptor is n... more The intrinsic protein tyrosine kinase activity of the epidermal growth factor (EGF) receptor is necessary for ligand-induced signaling. To determine whether cellular protein tyrosine kinases are substrates for EGF-activated receptors, phosphotyrosine-containing proteins were isolated from EGF-treated cells and assayed for tyrosine kinase activity using peptide substrates. A tyrosine kinase activity that is distinct from the EGF receptor was adsorbed to monoclonal anti-phosphotyrosine antibody columns and eluted with phenyl phosphate. Near-maximal tyrosine phosphorylation of this kinase occurred within 1 min of cell stimulation with an ED50 for EGF of 2.5 nM. The kinase was deactivated by incubation with purified CD45 tyrosine phosphatase in vitro, but activity could be restored by incubation with purified EGF receptor and Mn2+ ATP. These results suggest a cascade of tyrosine kinase signaling analogous to well characterized serine/threonine kinase cascades.
We have developed a method to study the primary sequence specificities of protein kinases by usin... more We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.
Cells expressing mutant epidermal growth factor (EGF) receptors have been used to study mechanism... more Cells expressing mutant epidermal growth factor (EGF) receptors have been used to study mechanisms through which EGF increases phospholipase C (PLC) activity. C-terminal truncation mutant EGF receptors are markedly impaired in their ability to increase inositol phosphate formation compared with wild-type EGF receptors. Mutation of the single tyrosine self-phosphorylation site at residue 992 to phenylalanine in an EGF receptor truncated at residue 1000 abolished the ability of EGF to increase inositol phosphate formation. C-terminal deletion mutant receptors that are impaired in their ability to increase inositol phosphate formation effectively phosphorylate PLC-gamma at the same tyrosine residues as do wild-type EGF receptors. EGF enhances PLC-gamma association with wild-type EGF receptors but not with mutant receptors lacking sites of tyrosine phosphorylation. These results indicate that formation of a complex between self-phosphorylated EGF receptors and PLC-gamma is necessary for...
Considerable effort is currently being devoted to understand the functions of protein p53, a majo... more Considerable effort is currently being devoted to understand the functions of protein p53, a major regulator of cell proliferation. The protein p53 has been reported to catalyse the annealing of complementary DNA or RNA strands. We report that this activity is inhibited in the presence of the serine/threonine protein kinase CK2. It is shown that this inhibition can be explained by the occurrence of a high-affinity molecular association between p53 and CK2. The molecular complex involves an interaction between the C-terminal domain of p53 and the β subunit of the oligomeric kinase. Accordingly, the isolated α subunit of the kinase was without effect. In addition, after phosphorylation by CK2, phosphorylated p53 lost its DNA annealing activity. Because the C-terminal domain of p53 is both involved in the association with CK2 and phosphorylated by it, our results suggest that either protein–protein interaction or phosphorylation of this domain might control the base pairing of compleme...
The Fas associated death domain protein (FADD) is the key adaptor molecule of the apoptotic signa... more The Fas associated death domain protein (FADD) is the key adaptor molecule of the apoptotic signal triggered by death receptors of the TNF-R1 superfamily. Besides its crucial role in the apoptotic machinery, FADD has proved to be important in many biological processes like tumorigenesis, embryonic development or cell cycle progression. In a process to decipher the regulatory mechanisms underlying FADD regulation, we identified the anti-apoptotic kinase, CK2, as a new partner and regulator of FADD sub-cellular localization. The blockade of CK2 activity induced FADD re-localization within the cell. Moreover, cytoplasmic FADD was increased when CK2β was knocked down. In vitro kinase and pull down assays confirmed that FADD could be phosphorylated by the CK2 holoenzyme. We found that phosphorylation is weak with CK2α alone and optimal in the presence of stoichiometric amounts of CK2α catalytic and CK2β regulatory subunits, showing that FADD phosphorylation is undertaken by the CK2 holoe...
A Molecular and Cellular View of Protein Kinase CK2, 1999
To date, the intracellular regulation of protein kinase CK2 is unknown. However it was observed t... more To date, the intracellular regulation of protein kinase CK2 is unknown. However it was observed that the enzyme associates with several intracellular proteins and the formation of such molecular complexes may represent a mechanism for the control of CK2 activity. Using the Interaction Trap system in yeast, with the CK2β as a bait, we looked for CK2 partners. We present the identification of new potential partners of CK2β and it is hoped that their classification will help in understanding the physiological roles and the regulation of CK2 in the cell.
A Molecular and Cellular View of Protein Kinase CK2, 1999
We have characterized several subdomains of the β subunit of protein kinase CK2. The N-terminal h... more We have characterized several subdomains of the β subunit of protein kinase CK2. The N-terminal half of the protein exhibits a pseudo-substrate segment in tandem with a polyamine binding domain responsible for the activation of the kinase by these polybasic compounds. Study of the chemical features of this polyamine binding site showed that polyamine analogs exhibiting the highest affinity for CK2 are the best CK2 activators. Mutational analysis disclosed that glutamic residues lying in the polyacidic region of the CK2β subunit are involved in the interaction with polyamine molecules and allowed the delineation of an autonomous binding domain. Furthermore, this regulatory domain was shown to mediate the association of CK2 with plasma membrane. The C-terminal domain of the CK2β subunit plays a role in the oligomerization of the kinase since it was observed that a truncated form of this subunit lacking its 33-last amino acids was incompetent for the assembly of polymeric forms of CK2. Altogether, our results support the notion that the β subunit of CK2 is a modular protein made by the association of interdependent domains that are involved in its multiple functions.
Protein Kinase CK2 — From Structure to Regulation, 2001
We have generated fusion proteins between the subunits of CK2 and GFP and characterized their beh... more We have generated fusion proteins between the subunits of CK2 and GFP and characterized their behaviour in living cells. The expressed fusion proteins were functional and interacted with endogenous CK2. Imaging of NIH3T3 cells expressing low level of GFP-CK2α or GFP-CK2β showed that both proteins were mostly nuclear in interphase. Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Once in the nucleus, both subunits diffused rapidly in the nucleoplasm. In mitotic cells, CK2 subunits were dispersed throughout the cytoplasm and were not associated to chromatin. Our data are compatible with the idea that each subunit can translocate individually to the nucleus to interact with each other or with important cellular partners. Understanding the molecular mechanisms which regulate the dynamic localization of CK2 subunits will be of central importance.
Protein kinase CK2 is a tetramer composed of two α catalytic subunits and two β regulatory subuni... more Protein kinase CK2 is a tetramer composed of two α catalytic subunits and two β regulatory subunits. The structure of a C-terminal truncated form of the human β subunit has been determined by X-ray crystallography to 1.7 Å resolution. One dimer is observed in the asymmetric unit of the crystal. The most striking feature of the structure is the presence of a zinc finger mediating the dimerization. The monomer structure consists of two domains, one entirely α-helical and one including the zinc finger. The dimer has a crescent shape holding a highly acidic region at both ends. We propose that this acidic region is involved in the interactions with the polyamines and/or catalytic subunits. Interestingly, conserved amino acid residues among β subunit sequences are clustered along one linear ridge that wraps around the entire dimer. This feature suggests that protein partners may interact with the dimer through a stretch of residues in an extended conformation.
Protein kinase CK2 is a multi-subunit complex whose dynamic assembly appears as a crucial point o... more Protein kinase CK2 is a multi-subunit complex whose dynamic assembly appears as a crucial point of regulation. The ability to interfere with specific protein-protein interactions has already provided powerful means of influencing the functions of selected proteins within the cell. CK2beta-derived cyclopeptides that target a well-defined hydrophobic pocket on CK2alpha have been previously characterized as potent inhibitors of CK2 subunit assembly [9]. As a first step toward the rational design of low molecular weight CK2 antagonists, we have in the present study screened a collection of podophyllotoxine indolo-analogues to identify chemical inhibitors of the CK2 subunit interaction. We report the identification of a podophyllotoxine indolo-analogue as a chemical ligand that binds to the CK2alpha/CK2beta interface inducing selective disruption of the CK2alpha/CK2beta assembly and concomitant inhibition of CK2alpha activity.
The Epstein-Barr Virus (EBV) early protein EB2 (also called BMLF1, Mta, or SM) promotes the nucle... more The Epstein-Barr Virus (EBV) early protein EB2 (also called BMLF1, Mta, or SM) promotes the nuclear export of a subset of early and late viral mRNAs and is essential for the production of infectious virions. We show here that in vitro, protein kinase CK2α and -β subunits bind both individually and, more efficiently, as a complex to the EB2 N terminus and that the CK2β regulatory subunit also interacts with the EB2 C terminus. Immunoprecipitated EB2 has CK2 activity that phosphorylates several sites within the 80 N-terminal amino acids of EB2, including Ser-55, -56, and -57, which are localized next to the nuclear export signal. EB2S3E, the phosphorylation-mimicking mutant of EB2 at these three serines, but not the phosphorylation ablation mutant EB2S3A, efficiently rescued the production of infectious EBV particles by HEK293 BMLF1-KO cells harboring an EB2-defective EBV genome. The defect of EB2S3A in transcomplementing 293 BMLF1-KO cells was not due to impaired nucleocytoplasmic sh...
1 The abbreviations used are: CK2, protein kinase CK2; MBP, maltose-binding protein; MBP51-110, ... more 1 The abbreviations used are: CK2, protein kinase CK2; MBP, maltose-binding protein; MBP51-110, fusion protein of MBP and the CK2  subunit domain Asp 51-Pro 110 ; MBP1-215, fusion protein of MBP and the entire CK2  subunit; MBP3, fusion protein of MBP and the entire CK2 Ala60,Ala61,Ala63 subunit; HA, fusion of the hemagglutinin tag at the C terminus of the CK2 regulatory subunit; 3HA, fusion of the hemagglutinin tag at the C terminus of the Ala60,Ala61,Ala63 subunit; CD, circular dichroism; PCR, polymerase chain reaction; BSA, bovine serum albumin.
The presence of fibroblast growth factor-2 (FGF-2) in the nucleus has now been reported both in v... more The presence of fibroblast growth factor-2 (FGF-2) in the nucleus has now been reported both in vitro and in vivo, but its nuclear functions are unknown. Here, we show that FGF-2 added to nuclear extract binds to protein kinase CK2 and nucleolin, a CK2 natural substrate. Added to baculovirus-infected cell extracts overexpressing CK2 or its isolated subunits, FGF-2 binds to the enzyme through its regulatory  subunit. Using purified proteins, FGF-2 is shown to directly interact with CK2 and to stimulate CK2 activity toward nucleolin. Furthermore, a mitogenic-deficient FGF-2 mutant protein has an impaired ability to interact with CK2 and to stimulate CK2 activity using nucleolin as substrate. We propose that in growing cells, one function of nuclear FGF-2 is to modulate CK2 activity through binding to its regulatory  subunit.
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Papers by O. Filhol