scholarly journals Complex Effects of Naturally Occurring Mutations in the JAK3 Pseudokinase Domain: Evidence for Interactions between the Kinase and Pseudokinase Domains

2000 ◽  
Vol 20 (3) ◽  
pp. 947-956 ◽  
Author(s):  
Min Chen ◽  
Alan Cheng ◽  
Fabio Candotti ◽  
Yong-Jie Zhou ◽  
Anka Hymel ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Intramolecular Interaction ◽  
Severe Combined Immunodeficiency ◽  
Kinase Domain ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT The structure of Janus kinases (JAKs) is unique among protein tyrosine kinases in having tandem, nonidentical kinase and pseudokinase domains. Despite its conservation in evolution, however, the function of the pseudokinase domain remains poorly understood. Lack of JAK3 expression results in severe combined immunodeficiency (SCID). In this study, we analyze two SCID patients with mutations in the JAK3 pseudokinase domain, which allows for protein expression but disrupts the regulation of the kinase activity. Specifically, these mutant forms of JAK3 had undetectable kinase activity in vitro but were hyperphosphorylated both in patients' Epstein-Barr virus-transformed B cells and when overexpressed in COS7 cells. Moreover, reconstitution of cells with these mutants demonstrated that, although they were constitutively phosphorylated basally, they were unable to transmit cytokine-dependent signals. Further analysis showed that the isolated catalytic domain of JAK3 was functional whereas either the addition of the pseudokinase domain or its deletion from the full-length molecule reduced catalytic activity. Through coimmunoprecipitation of the isolated pseudokinase domain with the isolated catalytic domain, we provide the first evidence that these two domains interact. Furthermore, whereas the wild-type pseudokinase domain modestly inhibited kinase domain-mediated STAT5 phosphorylation, the patient-derived mutants markedly inhibited this phosphorylation. We thus conclude that the JAK3 pseudokinase domain is essential for JAK3 function by regulating its catalytic activity and autophosphorylation. We propose a model in which this occurs via intramolecular interaction with the kinase domain and that increased inhibition of kinase activity by the pseudokinase domain likely contributes to the disease pathogenesis in these two patients.

scholarly journals Negative regulation of Raf-1 by phosphorylation of serine 621.

1996 ◽  
Vol 16 (10) ◽  
pp. 5409-5418 ◽  
Author(s):  
H Mischak ◽  
T Seitz ◽  
P Janosch ◽  
M Eulitz ◽  
H Steen ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Negative Regulation ◽  
Catalytic Function ◽  
Dependent Protein ◽  
The One

The elevation of cyclic AMP (cAMP) levels in the cell downregulates the activity of the Raf-1 kinase. It has been suggested that this effect is due to the activation of cAMP-dependent protein kinase (PKA), which can directly phosphorylate Raf-1 in vitro. In this study, we confirmed this hypothesis by coexpressing Raf-1 with the constitutively active catalytic subunit of PKA, which could fully reproduce the inhibition previously achieved by cAMP. PKA-phosphorylated Raf-1 exhibits a reduced affinity for GTP-loaded Ras as well as impaired catalytic activity. As the binding to GTP-loaded Ras induces Raf-1 activation in the cell, we examined which mechanism is required for PKA-mediated Raf-1 inhibition in vivo. A Raf-1 point mutant (RafR89L), which is unable to bind Ras, as well as the isolated Raf-1 kinase domain were still fully susceptible to inhibition by PKA, demonstrating that the phosphorylation of the Raf-1 kinase suffices for inhibition. By the use of mass spectroscopy and point mutants, PKA phosphorylation site was mapped to a single site in the Raf-1 kinase domain, serine 621. Replacement of serine 621 by alanine or cysteine or destruction of the PKA consensus motif by changing arginine 618 resulted in the loss of catalytic activity. Notably, a mutation of serine 619 to alanine did not significantly affect kinase activity or regulation by activators or PKA. Changing serine 621 to aspartic acid yielded a Raf-1 protein which, when expressed to high levels in Sf-9 insect cells, retained a very low inducible kinase activity that was resistant to PKA downregulation. The purified Raf-1 kinase domain displayed slow autophosphorylation of serine 621, which correlated with a decrease in catalytic function. The Raf-1 kinase domain activated by tyrosine phosphorylation could be downregulated by PKA. Specific removal of the phosphate residue at serine 621 reactivated the catalytic activity. These results are most consistent with a dual role of serine 621. On the one hand, serine 621 appears essential for catalytic activity; on the other hand, it serves as a phosphorylation site which confers negative regulation.

scholarly journals The common src homology region 2 domain of cytoplasmic signaling proteins is a positive effector of v-fps tyrosine kinase function.

1989 ◽  
Vol 9 (10) ◽  
pp. 4131-4140 ◽  
Author(s):  
C A Koch ◽  
M Moran ◽  
I Sadowski ◽  
T Pawson
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Specific Activity ◽  
Intramolecular Interaction ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Homology Region ◽  
Src Homology ◽  
Type V

A conserved noncatalytic domain SH2 (for src homology region 2) is located immediately N terminal to the kinase domains of all cytoplasmic protein-tyrosine kinases. We found that the wild-type v-fps SH2 domain stimulated the enzymatic activity of the adjacent kinase domain 10-fold and functioned as a powerful positive effector of catalytic and transforming activities within the v-fps oncoprotein (P130gag-fps). Partial proteolysis of P130gag-fps and supporting genetic data indicated that the v-fps SH2 domain exerts its effect on catalytic activity through an intramolecular interaction with the kinase domain. Amino acid alterations in the SH2 domain that impaired kinase function interfered with association of the SH2 domain with the kinase domain. Deletion of a conserved octapeptide motif converted the v-fps SH2 domain from an activator to an inhibitor of tyrosine kinase activity. This latent inhibitory activity of v-fps SH2 has functional implications for phospholipase C-gamma and p21ras GTPase-activating protein, both of which have two distinct SH2 domains suggestive of complex regulation. In addition to regulating the specific activity of the kinase domain, the SH2 domain of P130gag-fps was also found to be required for the tyrosine phosphorylation of specific cellular proteins, notably polypeptides of 124 and 62 kilodaltons. The SH2 domain therefore appears to play a dual role in regulation of kinase activity and recognition of cellular substrates.

scholarly journals A Cyclin-Binding Motif within the Amino-Terminal Homology Domain of EBNA3C Binds Cyclin A and Modulates Cyclin A-Dependent Kinase Activity in Epstein-Barr Virus-Infected Cells

2004 ◽  
Vol 78 (23) ◽  
pp. 12857-12867 ◽  
Author(s):  
Jason S. Knight ◽  
Nikhil Sharma ◽  
Danielle E. Kalman ◽  
Erle S. Robertson
Keyword(s):  
Amino Acids ◽  
Epstein Barr Virus ◽  
Kinase Activity ◽  
Cyclin A ◽  
Carboxy Terminus ◽  
Barr Virus ◽  
Amino Terminal ◽  
Infected Cells ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is a virus-encoded latent antigen essential for primary B-cell transformation. In this report we demonstrate that although the carboxy terminus of EBNA3C predominantly regulates cyclin A-dependent kinase activity, the region of greatest affinity for cyclin A lies within the EBNA3 amino-terminal homology domain of EBNA3C. Detailed mapping studies employing both in vitro binding assays and coimmunoprecipitation experiments implicated a small region of EBNA3C, amino acids 130 to 159 within the EBNA3 homology domain, as having the greatest affinity for cyclin A. The EBNA3 homology domain has the highest degree of amino acid similarity (approximately 30%) between the EBNA3 proteins, and, indeed, EBNA3B, but not EBNA3A, showed binding activity with cyclin A. We also show that EBNA3C binds to the α1 helix of the highly conserved mammalian cyclin box, with cyclin A amino acids 206 to 226 required for strong binding to EBNA3C amino acids 130 to 159. Interestingly, EBNA3C also bound human cyclins D1 and E in vitro, although the affinity was approximately 30% of that seen for cyclin A. Previously it was demonstrated that full-length EBNA3C rescues p27-mediated suppression of cyclin A-dependent kinase activity (J. S. Knight and E. S. Robertson, J. Virol. 78:1981-1991, 2004). It was also demonstrated that the carboxy terminus of EBNA3C recapitulates this phenotype. Surprisingly, the amino terminus of EBNA3C with the highest affinity for cyclin A was unable to rescue p27 suppression of kinase activity and actually downregulates cyclin A activity when introduced into EBV-infected cells. The data presented here suggests that the amino terminus of EBNA3C may play an important role in recruiting cyclin A complexes, while the carboxy terminus of EBNA3C is necessary for the functional modulation of cyclin A complex kinase activity.

The common src homology region 2 domain of cytoplasmic signaling proteins is a positive effector of v-fps tyrosine kinase function

1989 ◽  
Vol 9 (10) ◽  
pp. 4131-4140
Author(s):  
C A Koch ◽  
M Moran ◽  
I Sadowski ◽  
T Pawson
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Specific Activity ◽  
Intramolecular Interaction ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Homology Region ◽  
Src Homology ◽  
Type V

A conserved noncatalytic domain SH2 (for src homology region 2) is located immediately N terminal to the kinase domains of all cytoplasmic protein-tyrosine kinases. We found that the wild-type v-fps SH2 domain stimulated the enzymatic activity of the adjacent kinase domain 10-fold and functioned as a powerful positive effector of catalytic and transforming activities within the v-fps oncoprotein (P130gag-fps). Partial proteolysis of P130gag-fps and supporting genetic data indicated that the v-fps SH2 domain exerts its effect on catalytic activity through an intramolecular interaction with the kinase domain. Amino acid alterations in the SH2 domain that impaired kinase function interfered with association of the SH2 domain with the kinase domain. Deletion of a conserved octapeptide motif converted the v-fps SH2 domain from an activator to an inhibitor of tyrosine kinase activity. This latent inhibitory activity of v-fps SH2 has functional implications for phospholipase C-gamma and p21ras GTPase-activating protein, both of which have two distinct SH2 domains suggestive of complex regulation. In addition to regulating the specific activity of the kinase domain, the SH2 domain of P130gag-fps was also found to be required for the tyrosine phosphorylation of specific cellular proteins, notably polypeptides of 124 and 62 kilodaltons. The SH2 domain therefore appears to play a dual role in regulation of kinase activity and recognition of cellular substrates.

Structural Positioning of the SH2 Domain Is Critical for Bcr-Abl Kinase Activity, Signal Transduction and Oncogenic Transformation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 569-569
Author(s):  
Oliver D. Hantschel ◽  
Florian Grebien ◽  
Ines Kaupe ◽  
Giulio Superti-Furga
Keyword(s):  
Signal Transduction ◽  
Catalytic Activity ◽  
Kinase Activity ◽  
Critical Factor ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Pharmacological Intervention ◽  
Single Point Mutation ◽  
Mouse Bone Marrow

Abstract We have recently shown that the SH2 domain stimulates c-Abl catalytic activity and substrate phosphorylation. This effect is exerted directly through the establishment of a tight SH2-kinase domain interface in the active conformation of c-Abl (Filippakopoulos et al. (2008) Cell, in press, scheduled to be published on September 5, 2008). Mutations in the SH2 domain that presumably disrupt this SH2-kinase domain interface, such as the Ile164Glu mutation, result in severe impairment of Abl catalytic activity. Thus, correct positioning of the SH2 and kinase domain modules appears to be critical for efficient activation of cytoplasmic tyrosine kinases. Here, we present data showing that the same structural coupling of the SH2 and kinase domain is also a critical factor for full activation of the oncogenic fusion kinase Bcr-Abl. A single point mutation in the SH2 domain (Ile164Glu) led to a dramatic reduction in Bcr-Abl in vitro tyrosine kinase activity and Bcr-Abl autophosphorylation, both on the activation loop (pTyr-412) and the SH2-kinase domain linker (pTyr-245). This resulted in a strong decrease in global cellular tyrosine phosphorylation, as well as decreased phosphorylation of critical downstream mediators of Bcr-Abl signaling. Both wildtype Bcr-Abl, as well as the Bcr-Abl Ile164Glu mutant were able to confer factor independent growth to Ba/F3- and UT-7 cell lines, although to a different extent. Detailed data on the properties of the Ile164Glu mutation in vitro, in imatinib inhibition assays, transformation assays and mouse bone marrow transplant models will be presented. We propose that the structural positioning of the SH2 domain is a crucial factor for constitutive activity, signal transduction and transforming capacity of Bcr-Abl. Besides oligomerization via the N-terminal coiled-coiled domain and loss of the auto-inhibitory N-terminal myristoyl group, the proper positioning of the SH2 domain appears to be another critical factor that is required for constitutive activation of Bcr- Abl, which is the prerequisite for its ability to induce chronic myeloid leukemia (CML). Inhibitors of the SH2-kinase domain interface of Bcr-Abl may comprise alternative or additional points of pharmacological intervention for the treatment of imatinib-sensitive or -resistant CML or Ph+ acute lymphocytic leukemia.

scholarly journals Regulation of Sos Activity by Intramolecular Interactions

1998 ◽  
Vol 18 (2) ◽  
pp. 880-886 ◽  
Author(s):  
Senena Corbalan-Garcia ◽  
Steluta M. Margarit ◽  
Dalia Galron ◽  
Shao-song Yang ◽  
Dafna Bar-Sagi
Keyword(s):  
Catalytic Activity ◽  
Tyrosine Kinases ◽  
Catalytic Domain ◽  
Intramolecular Interactions ◽  
Carboxyl Terminus ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Exchange Activity

ABSTRACT The guanine nucleotide exchange factor Sos mediates the coupling of receptor tyrosine kinases to Ras activation. To investigate the mechanisms that control Sos activity, we have analyzed the contribution of various domains to its catalytic activity. Using human Sos1 (hSos1) truncation mutants, we show that Sos proteins lacking either the amino or the carboxyl terminus domain, or both, display a guanine nucleotide exchange activity that is significantly higher compared with that of the full-length protein. These results demonstrate that both the amino and the carboxyl terminus domains of Sos are involved in the negative regulation of its catalytic activity. Furthermore, in vitro Ras binding experiments suggest that the amino and carboxyl terminus domains exert negative allosteric control on the interaction of the Sos catalytic domain with Ras. The guanine nucleotide exchange activity of hSos1 was not augmented by growth factor stimulation, indicating that Sos activity is constitutively maintained in a downregulated state. Deletion of both the amino and the carboxyl terminus domains was sufficient to activate the transforming potential of Sos. These findings suggest a novel negative regulatory role for the amino terminus domain of Sos and indicate a cooperation between the amino and the carboxyl terminus domains in the regulation of Sos activity.

scholarly journals Epstein-Barr Virus Nuclear Antigen 3C Regulates Cyclin A/p27 Complexes and Enhances Cyclin A-Dependent Kinase Activity

2004 ◽  
Vol 78 (4) ◽  
pp. 1981-1991 ◽  
Author(s):  
Jason S. Knight ◽  
Erle S. Robertson
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Cell Transformation ◽  
Cyclin A ◽  
Nuclear Antigen ◽  
Cycle Progression ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is essential for primary B-cell transformation. In this report we show that cyclin A, an activator of S phase progression, bound tightly to EBNA3C. EBNA3C interacted with cyclin A in vitro and associated with cyclin A complexes in EBV-transformed lymphoblastoid cell lines. Importantly, EBNA3C stimulated cyclin A-dependent kinase activity and rescued p27-mediated inhibition of cyclin A/Cdk2 kinase activity by decreasing the molecular association between cyclin A and p27 in cells. Additionally, phosphorylation of the retinoblastoma protein, a major regulator of cell cycle progression, was enhanced both in vitro and in vivo in the presence of EBNA3C. Cyclin A interacted with a region of the carboxy terminus of EBNA3C, shown to be important both for stimulation of cyclin A-dependent kinase activity and for cell cycle progression. This provides the first evidence of an essential EBV latent antigen's directly targeting a cell cycle regulatory protein and suggests a novel mechanism by which EBV deregulates the mammalian cell cycle, which is of critical importance in B-cell transformation.

scholarly journals Activation of Jak2 catalytic activity requires phosphorylation of Y1007 in the kinase activation loop.

1997 ◽  
Vol 17 (5) ◽  
pp. 2497-2501 ◽  
Author(s):  
J Feng ◽  
B A Witthuhn ◽  
T Matsuda ◽  
F Kohlhuber ◽  
I M Kerr ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Kinase Activity ◽  
Critical Role ◽  
Regulatory Region ◽  
Kinase Domain ◽  
Detectable Effect ◽  
Activation Loop ◽  
Receptor Complexes ◽  
Induced Aggregation

The Janus protein tyrosine kinases (Jaks) play critical roles in transducing growth and differentiation signals emanating from ligand-activated cytokine receptor complexes. The activation of the Jaks is hypothesized to occur as a consequence of auto- or transphosphorylation on tyrosine residues associated with ligand-induced aggregation of the receptor chains and the associated Jaks. In many kinases, regulation of catalytic activity by phosphorylation occurs on residues within the activation loop of the kinase domain. Within the Jak2 kinase domain, there is a region that has considerable sequence homology to the regulatory region of the insulin receptor and contains two tyrosines, Y1007 and Y1008, that are potential regulatory sites. In the studies presented here, we demonstrate that among a variety of sites, Y1007 and Y1008 are sites of trans- or autophosphorylation in vivo and in in vitro kinase reactions. Mutation of Y1007, or both Y1007 and Y1008, to phenylalanine essentially eliminated kinase activity, whereas mutation of Y1008 to phenylalanine had no detectable effect on kinase activity. The mutants were also examined for the ability to reconstitute erythropoietin signaling in gamma2 cells, which lack Jak2. Consistent with the kinase activity, mutation of Y1007 to phenylalanine eliminated the ability to restore signaling. Moreover, phosphorylation of a kinase-inactive mutant (K882E) was not detected, indicating that Jak2 activation during receptor aggregation is dependent on Jak2 and not another receptor-associated kinase. The results demonstrate the critical role of phosphorylation of Y1007 in Jak2 regulation and function.

scholarly journals A Protein Kinase Activity Associated with Epstein-Barr Virus BGLF4 Phosphorylates the Viral Early Antigen EA-D In Vitro

2000 ◽  
Vol 74 (7) ◽  
pp. 3093-3104 ◽  
Author(s):  
Mei-Ru Chen ◽  
Shin-Jye Chang ◽  
Hsiaowen Huang ◽  
Jen-Yang Chen
Keyword(s):  
Protein Kinase ◽  
Epstein Barr Virus ◽  
Kinase Activity ◽  
Kinase Assay ◽  
Specific Antiserum ◽  
Protein Kinase Activity ◽  
Early Antigen ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) open reading frame BGLF4 was identified as a potential Ser/Thr protein kinase gene through the recognition of amino acid sequence motifs characteristic of conserved regions within the catalytic domains of protein kinases. In order to investigate this potential kinase activity, BGLF4 was expressed inEscherichia coli and the purified protein was used to generate a specific antiserum. Recombinant vaccinia virus vTF7-3, which expresses the T7 RNA polymerase, was used to infect 293 and 293T cells after transient transfection with a plasmid containing BGLF4 under the control of the T7 promoter. Autophosphorylation of the BGLF4 protein was demonstrated using the specific antiserum in an immune complex kinase assay. In addition, EBNA-1-tagged BGLF4 and EBNA-1 monoclonal antibody 5C11 were used to demonstrate the specificity of the kinase activity and to locate BGLF4 in the cytoplasm of transfected cells. Manganese ions were found to be essential for autophosphorylation of BGLF4, and magnesium can stimulate the activity. BGLF4 can utilize GTP, in addition to ATP, as a phosphate donor in this assay. BGLF4 can phosphorylate histone and casein in vitro. Among the potential viral protein substrates we examined, the EBV early antigen (EA-D, BMRF1), a DNA polymerase accessory factor and an important transactivator during lytic infection, was found to be phosphorylated by BGLF4 in vitro. Amino acids 1 to 26 of BGLF4, but not the predicted conserved catalytic domain, were found to be essential for autophosphorylation of BGLF4.

scholarly journals Interleukin-1β Expression in Human Gastric Carcinoma with Epstein-Barr Virus Infection

2002 ◽  
Vol 76 (13) ◽  
pp. 6825-6831 ◽  
Author(s):  
Ja-Mun Chong ◽  
Kazuya Sakuma ◽  
Makoto Sudo ◽  
Toshio Osawa ◽  
Etsuko Ohara ◽  
...  
Keyword(s):  
Gastric Carcinoma ◽  
Epstein Barr Virus ◽  
Severe Combined Immunodeficiency ◽  
Interleukin 1Β ◽  
Oligonucleotide Array ◽  
Enzyme Linked Immunosorbent Assay ◽  
Epstein Barr Virus Infection ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT The KT tumor is a transplantable strain of a human Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC), established in severe combined immunodeficiency (SCID) mice, with which the cytokine expression of EBVaGC can be investigated without interference from the infiltrating lymphocytes. As a part of a high-density oligonucleotide array (GeneChip) analysis of EBVaGC, the interleukin-1β (IL-1β) gene was the only cytokine gene that showed markedly higher expression in the KT tumor cells than in two tumor strains of EBV-negative GC. The results were confirmed by Northern blotting, Western blotting, and enzyme-linked immunosorbent assay. Furthermore, we demonstrated a positive signal for IL-1β mRNA in the carcinoma cells of a surgically resected EBVaGC, but not in EBV-negative GC, by in situ hybridization. In vitro, IL-1β increased the cell growth of a GC cell line, TMK1. Thus, IL-1β may act as an autocrine growth factor in EBVaGC.

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