scholarly journals Site-directed mutagenesis of Lys-174, Asp-179 and Asp-191 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

1997 ◽  
Vol 321 (3) ◽  
pp. 623-627 ◽  
Author(s):  
Luc BERTRAND ◽  
Johan DEPREZ ◽  
Didier VERTOMMEN ◽  
Attilio DI PIETRO ◽  
Louis HUE ◽  
...  
Keyword(s):  
Adenylate Kinase ◽  
Kinase Activity ◽  
Structural Model ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Structure Model ◽  
Phosphate Binding ◽  
Fluorescence Measurements ◽  
Natural Mutation ◽  
Kinase Structure

In a structural model of the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase based on the analogy with adenylate kinase, Lys-174, Asp-179 and Asp-191 residues are located in the putative active site. Asp-179 and Asp-191 are conserved in all known 6-phosphofructo-2-kinase sequences. In contrast, Lys-174 is conserved except in a yeast isoenzyme, fbp26, where it is replaced by glycine. Yeast fbp26 possesses fructose-2,6-bisphosphatase activity, but is devoid of 6-phosphofructo-2-kinase activity. Mutation of Asp-179 and Asp-191 of the rat liver isoenzyme to alanine increased the Km of 6-phosphofructo-2-kinase for fructose 6-phosphate 2000-and 1000-fold respectively, whereas mutation of Lys-174 to glycine decreased the Vmax of 6-phosphofructo-2-kinase more than 4000-fold. In contrast, none of the mutations affected the kinetic parameters of fructose-2,6-bisphosphatase. CD and fluorescence measurements indicated that the mutations had no effect on the structure and stability of the recombinant proteins. The results show that Asp-179 and Asp-191 participate in fructose 6-phosphate binding, whereas Lys-174 is important for catalysis. Therefore the natural mutation of Lys-174 to glycine in the fbp26 yeast isoenzyme could explain the lack of 6-phosphofructo-2-kinase activity. These results support a novel 6-phosphofructo-2-kinase structure model based on adenylate kinase.

scholarly journals Mutagenesis of charged residues in a conserved sequence in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

1997 ◽  
Vol 321 (3) ◽  
pp. 609-614 ◽  
Author(s):  
Luc BERTRAND ◽  
Didier VERTOMMEN ◽  
Ernest FEYTMANS ◽  
Attilio Di PIETRO ◽  
Mark H. RIDER ◽  
...  
Keyword(s):  
Structural Changes ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Magnesium Citrate ◽  
Phosphate Binding ◽  
Conserved Sequence ◽  
Kinase Mutation

Arg-136, Glu-137, Arg-138 and Arg-139 are conserved in all sequences of the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Their role was studied by site-directed mutagenesis. All the mutations had little, if any, effect on fructose-2,6-bisphosphatase activity. Mutations of Arg-136 and Glu-137 into Ala caused only minor modifications of phosphofructo-2-kinase activity. In contrast, mutation of Arg-138 into Ala increased 280-fold the Km for fructose 6-phosphate of phosphofructo-2-kinase. Mutation of Arg-139 into Ala resulted in decreases in phosphofructo-2-kinase Vmax/Km for MgATP and fructose 6-phosphate 600-fold and 5000-fold respectively. Mutation of Arg-139 into Lys and Gln increased the Km of phosphofructo-2-kinase for MgATP (20-fold and 25-fold respectively) and for fructose 6-phosphate (8-fold and 13-fold), and the IC50 for MgADP (30-fold and 50-fold) and for magnesium citrate (7-fold and 25-fold). However, these two mutations did not affect nucleotide binding, as measured by quenching of intrinsic fluorescence. The changes in kinetic properties induced by mutations could not be attributed to structural changes. It is proposed that Arg-138 is involved in fructose 6-phosphate binding and that Arg-139 is probably involved in the stabilization of the transition state and so participates in catalysis.

scholarly journals Modelling the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase on adenylate kinase

1997 ◽  
Vol 321 (3) ◽  
pp. 615-621 ◽  
Author(s):  
Luc BERTRAND ◽  
Didier VERTOMMEN ◽  
Eric DEPIEREUX ◽  
Louis HUE ◽  
Mark H. RIDER ◽  
...  
Keyword(s):  
Adenylate Kinase ◽  
Structural Model ◽  
Three Dimensional ◽  
Kinase Domain ◽  
Multiple Alignment ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Conserved Residues ◽  
Nucleotide Binding Proteins ◽  
Sequence Similarities

Simultaneous multiple alignment of available sequences of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase revealed several segments of conserved residues in the 2-kinase domain. The sequence of the kinase domain was also compared with proteins of known three-dimensional structure. No similarity was found between the kinase domain of 6-phosphofructo-2-kinase and 6-phosphofructo-1-kinase. This questions the modelling of the 2-kinase domain on bacterial 6-phosphofructo-1-kinase that has previously been proposed [Bazan, Fletterick and Pilkis (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642Ő9646]. However, sequence similarities were found between the 2-kinase domain and several nucleotide-binding proteins, the most similar being adenylate kinase. A structural model of the 2-kinase domain based on adenylate kinase is proposed. It accommodates all the results of site-directed mutagenesis studies carried out to date on residues in the 2-kinase domain. It also allows residues potentially involved in catalysis and/or substrate binding to be predicted.

Site-directed mutagenesis of the SH2- and SH3-coding domains of c-src produces varied phenotypes, including oncogenic activation of p60c-src

1990 ◽  
Vol 10 (4) ◽  
pp. 1307-1318
Author(s):  
H Hirai ◽  
H E Varmus
Keyword(s):  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Point Mutations ◽  
Kinase Domain ◽  
Biological Properties ◽  
Site Directed Mutagenesis ◽  
Mutant Proteins ◽  
Positive Effects ◽  
Src Gene ◽  
Carboxy Terminal

The products of the viral and cellular src genes, p60v-src and p60c-src, appear to be composed of multiple functional domains. Highly conserved regions called src homology 2 and 3 (SH2 and SH3), comprising amino acid residues 88 to 250, are believed to modulate the protein-tyrosine kinase activity present in the carboxy-terminal halves of the src proteins. To explore the functions of these regions more fully, we have made 34 site-directed mutations in a transformation-competent c-src gene encoding phenylalanine in place of tyrosine 527 (Y527F c-src). Twenty of the new mutations change only one or two amino acids, and the remainder delete small or large portions of the SH2-SH3 region. These mutant alleles have been incorporated into a replication-competent Rous sarcoma virus vector to examine the biochemical and biological properties of the mutant proteins after infection of chicken embryo fibroblasts. Four classes of mutant proteins were observed: class 1, mutants with only slight differences from the parental gene products; class 2, mutant proteins with diminished transforming and specific kinase activities; class 3, mutant proteins with normal or enhanced specific kinase activity but impaired biological activity, often as a consequence of instability; and class 4, mutant proteins with augmented biological and catalytic activities. In general, there was a strong correlation between total kinase activity (or amounts of intracellular phosphotyrosine-containing proteins) and transforming activity. Deletion mutations and some point mutations affecting residues 109 to 156 inhibited kinase and transforming functions, whereas deletions affecting residues 187 to 226 generally had positive effects on one or both of those functions, confirming that SH2-SH3 has complex regulatory properties. Five mutations that augmented the transforming and kinase activities of Y527F c-src [F172P, R175L, delta(198-205), delta(206-226), and delta(176-226)] conferred transformation competence on an otherwise normal c-src gene, indicating that mutations in SH2 (like previously described lesions in SH3, the kinase domain, and a carboxy-terminal inhibitory domain) can activate c-src.

scholarly journals The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity

2021 ◽  
Vol 153 (6) ◽  
Author(s):  
Hana Inoue ◽  
Takashi Murayama ◽  
Takuya Kobayashi ◽  
Masato Konishi ◽  
Utako Yokoyama
Keyword(s):  
Oxidative Stress ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Full Length ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Zinc Binding ◽  
Binding Motif ◽  
Channel Activity ◽  
Zinc Binding Motif

The activity of the TRPM7 channel is negatively regulated by intracellular Mg2+. We previously reported that oxidative stress enhances the inhibition of TRPM7 by intracellular Mg2+. Here, we aimed to clarify the mechanism underlying TRPM7 inhibition by hydrogen peroxide (H2O2). Site-directed mutagenesis of full-length TRPM7 revealed that none of the cysteines other than C1809 and C1813 within the zinc-binding motif of the TRPM7 kinase domain were involved in the H2O2-induced TRPM7 inhibition. Mutation of C1809 or C1813 prevented expression of full-length TRPM7 on the plasma membrane. We therefore developed an assay to functionally reconstitute full-length TRPM7 by coexpressing the TRPM7 channel domain (M7cd) and the TRPM7 kinase domain (M7kd) as separate proteins in HEK293 cells. When M7cd was expressed alone, the current was inhibited by intracellular Mg2+ more strongly than that of full-length TRPM7 and was insensitive to oxidative stress. Coexpression of M7cd and M7kd attenuated the inhibition by intracellular Mg2+ and restored sensitivity to oxidative stress, indicating successful reconstitution of a full-length TRPM7-like current. We observed a similar effect when M7cd was coexpressed with the kinase-inactive mutant M7kd-K1645R, suggesting that the kinase activity is not essential for the reconstitution. However, coexpression of M7cd and M7kd carrying a mutation at either C1809 or C1813 failed to restore the full-length TRPM7-like current. No reconstitution was observed when using M7kd carrying a mutation at H1750 and H1807, which are involved in the zinc-binding motif formation with C1809 and C1813. These data suggest that the zinc-binding motif is essential for the intracellular Mg2+-dependent regulation of the TRPM7 channel activity by its kinase domain and that the cysteines in the zinc-binding motif play a role in the oxidative stress response of TRPM7.

First model of dimeric LRRK2: the challenge of unrevealing the structure of a multidomain Parkinson's-associated protein

2016 ◽  
Vol 44 (6) ◽  
pp. 1635-1641 ◽  
Author(s):  
Giambattista Guaitoli ◽  
Bernd K. Gilsbach ◽  
Francesco Raimondi ◽  
Christian Johannes Gloeckner
Keyword(s):  
Kinase Activity ◽  
Structural Model ◽  
Three Dimensional ◽  
Kinase Domain ◽  
Rational Drug Design ◽  
Dimensional Structure ◽  
Rational Drug ◽  
Its Gene ◽  
Lrrk2 Gene ◽  
Successful Approach

Mutations within the leucine-rich repeat kinase 2 (LRRK2) gene represent the most common cause of Mendelian forms of Parkinson's disease, among autosomal dominant cases. Its gene product, LRRK2, is a large multidomain protein that belongs to the Roco protein family exhibiting GTPase and kinase activity, with the latter activity increased by pathogenic mutations. To allow rational drug design against LRRK2 and to understand the cross-regulation of the G- and the kinase domain at a molecular level, it is key to solve the three-dimensional structure of the protein. We review here our recent successful approach to build the first structural model of dimeric LRRK2 by an integrative modeling approach.

Identification of dimer interactions required for the catalytic activity of the TRPM7 alpha-kinase domain

2009 ◽  
Vol 420 (1) ◽  
pp. 115-122 ◽  
Author(s):  
Scott W. Crawley ◽  
Graham P. Côté
Keyword(s):  
Transient Receptor Potential ◽  
Kinase Activity ◽  
Glutathione Transferase ◽  
Receptor Potential ◽  
Kinase Domain ◽  
Terminal Segment ◽  
Site Directed Mutagenesis ◽  
Dimer Formation ◽  
Transient Receptor Potential Melastatin ◽  
Activation Sequence

TRPM7 (transient receptor potential melastatin) combines an ion channel domain with a C-terminal protein kinase domain that belongs to the atypical alpha-kinase family. The TRPM7 alpha-kinase domain assembles into a dimer through the exchange of an N-terminal segment that extends from residue 1551 to residue 1577 [Yamaguchi, Matsushita, Nairn and Kuriyan (2001) Mol. Cell 7, 1047–1057]. Here, we show, by analysis of truncation mutants, that residues 1553–1562 of the N-terminus are essential for kinase activity but not dimer formation. Within this ‘activation sequence’, site-directed mutagenesis identified Tyr-1553 and Arg-1558 as residues critical for activity. Examination of the TRPM7 kinase domain structure suggests that the activation sequence interacts with the other subunit to help position a catalytic loop that contains the invariant Asp-1765 residue. Residues 1563–1570 of the N-terminal segment are critical for dimer assembly. Mutation of Leu-1564, Ile-1568 or Phe-1570 to alanine abolished both kinase activity and dimer formation. The activity of a monomeric TRPM7 kinase domain lacking the entire N-terminal segment was rescued by a GST (glutathione transferase) fusion protein containing residues 1548–1576 of TRPM7, showing that all interactions essential for activity are provided by the N-terminal segment. Activity was also restored by GST fused to the N-terminal segment of TRPM6 (residues 1711–1740), demonstrating the feasibility of forming functional TRPM6–TRPM7 alpha-kinase domain heterodimers. It is proposed that covalent modifications or binding interactions that alter the conformation of the N-terminal exchanged segment may provide a means to regulate TRPM7 kinase activity.

scholarly journals Site-directed mutagenesis of the SH2- and SH3-coding domains of c-src produces varied phenotypes, including oncogenic activation of p60c-src.

1990 ◽  
Vol 10 (4) ◽  
pp. 1307-1318 ◽  
Author(s):  
H Hirai ◽  
H E Varmus
Keyword(s):  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Point Mutations ◽  
Kinase Domain ◽  
Biological Properties ◽  
Site Directed Mutagenesis ◽  
Mutant Proteins ◽  
Positive Effects ◽  
Src Gene ◽  
Carboxy Terminal

The products of the viral and cellular src genes, p60v-src and p60c-src, appear to be composed of multiple functional domains. Highly conserved regions called src homology 2 and 3 (SH2 and SH3), comprising amino acid residues 88 to 250, are believed to modulate the protein-tyrosine kinase activity present in the carboxy-terminal halves of the src proteins. To explore the functions of these regions more fully, we have made 34 site-directed mutations in a transformation-competent c-src gene encoding phenylalanine in place of tyrosine 527 (Y527F c-src). Twenty of the new mutations change only one or two amino acids, and the remainder delete small or large portions of the SH2-SH3 region. These mutant alleles have been incorporated into a replication-competent Rous sarcoma virus vector to examine the biochemical and biological properties of the mutant proteins after infection of chicken embryo fibroblasts. Four classes of mutant proteins were observed: class 1, mutants with only slight differences from the parental gene products; class 2, mutant proteins with diminished transforming and specific kinase activities; class 3, mutant proteins with normal or enhanced specific kinase activity but impaired biological activity, often as a consequence of instability; and class 4, mutant proteins with augmented biological and catalytic activities. In general, there was a strong correlation between total kinase activity (or amounts of intracellular phosphotyrosine-containing proteins) and transforming activity. Deletion mutations and some point mutations affecting residues 109 to 156 inhibited kinase and transforming functions, whereas deletions affecting residues 187 to 226 generally had positive effects on one or both of those functions, confirming that SH2-SH3 has complex regulatory properties. Five mutations that augmented the transforming and kinase activities of Y527F c-src [F172P, R175L, delta(198-205), delta(206-226), and delta(176-226)] conferred transformation competence on an otherwise normal c-src gene, indicating that mutations in SH2 (like previously described lesions in SH3, the kinase domain, and a carboxy-terminal inhibitory domain) can activate c-src.

NPM-ALK Mutants in Kinase Domain Exhibit Altered Kinase Activity and Various Sensitivity to ALK Inhibitors.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1603-1603
Author(s):  
Lihui Lu ◽  
Arup Ghose ◽  
Matt Quail ◽  
Bruce Ruggeri ◽  
Mangeng Cheng
Keyword(s):  
Growth Inhibition ◽  
Kinase Activity ◽  
Mutant Cell ◽  
Single Point ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Alk Inhibitors ◽  
Anaplastic Lymphoma ◽  
Alk Inhibitor ◽  
In Cells

Abstract Abnormal expression of constitutively active anaplastic lymphoma kinase chimeric proteins (NPM-ALK) in the pathogenesis of anaplastic large-cell lymphoma (ALCL) is well defined. Recently, small molecule inhibitors have been reported that provide solid proof-of-concept validation that inhibition of ALK is sufficient to attenuate the growth and proliferation of ALK+ALCL cells. Although robust clinical response of ALCL patients to the ALK inhibitors is expected, some of those patients are also anticipated to develop resistance to an ALK inhibitor, most likely associated with single point mutations in the kinase domain of ALK. In this study, the nonsense mutants of NPM-ALK on the phosphate anchor region and the gatekeeper region were generated by site-directed mutagenesis and their kinase activity was measured in cells. NPM-ALK/L182M, L182V and L256M mutants displayed comparable or higher kinase activity in cells relative to NPM-ALK wt, and were able to render BaF3 cells into growth factor-independent growth, while NPM-ALK/L182R, L256R, L256V, L256P and L256Q displayed much weaker or little kinase activity in cells and failed to transform BaF3 cells. While NPM-ALK/L182M and L182V displayed comparable sensitivity to a fused pyrrolocarbazole (FP)-derived ALK inhibitor as NPM-ALK wt, they were >30-fold less sensitive to a diaminopyrimidine (DAP)-derived ALK inhibitor. On the other hand, NPM-ALK/L256M displayed >30-fold less sensitivity to both the FP-derived and the DAP-derived ALK inhibitors. Consistent with NPM-ALK autophosphorylation inhibition, the FP-ALK inhibitor induced growth inhibition and cytotoxicity of BaF3/NPM-ALK/L182M and L182V cells but not L256M cells, and the DAP ALK inhibitor failed to induce growth inhibition and cytotoxicity of all three BaF3/NPM-ALK mutant cell lines in culture. In the absence of an ALK inhibitor, BaF3 cells harboring NPM-ALK mutants did not display growth advantage in culture with 10% serum and in mice over the NPM-ALK wt cells. However, the BaF3/NPM-ALK mutants displayed significant growth advantage in culture with low serum (2%) over the BaF3/NPM-ALKwt cells, suggesting the BAF3/NPM-ALK mutant cells may proliferate better in the stressed conditions. Analyses of binding of ALK inhibitors to ALK wt and mutants in ALK homology models are in progress and the results will be discussed.

scholarly journals Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts

2016 ◽  
Vol 113 (30) ◽  
pp. E4357-E4366 ◽  
Author(s):  
Giambattista Guaitoli ◽  
Francesco Raimondi ◽  
Bernd K. Gilsbach ◽  
Yacob Gómez-Llorente ◽  
Egon Deyaert ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Structural Model ◽  
3D Structure ◽  
Kinase Domain ◽  
Full Length ◽  
Regulatory Domains ◽  
Catalytic Domains ◽  
Structural Framework ◽  
Kinase Domain Mutations

Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein containing two catalytic domains: a Ras of complex proteins (Roc) G-domain and a kinase domain. Mutations associated with familial and sporadic Parkinson’s disease (PD) have been identified in both catalytic domains, as well as in several of its multiple putative regulatory domains. Several of these mutations have been linked to increased kinase activity. Despite the role of LRRK2 in the pathogenesis of PD, little is known about its overall architecture and how PD-linked mutations alter its function and enzymatic activities. Here, we have modeled the 3D structure of dimeric, full-length LRRK2 by combining domain-based homology models with multiple experimental constraints provided by chemical cross-linking combined with mass spectrometry, negative-stain EM, and small-angle X-ray scattering. Our model reveals dimeric LRRK2 has a compact overall architecture with a tight, multidomain organization. Close contacts between the N-terminal ankyrin and C-terminal WD40 domains, and their proximity—together with the LRR domain—to the kinase domain suggest an intramolecular mechanism for LRRK2 kinase activity regulation. Overall, our studies provide, to our knowledge, the first structural framework for understanding the role of the different domains of full-length LRRK2 in the pathogenesis of PD.

scholarly journals Site-directed mutagenesis of rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: role of Asp-130 in the 2-kinase domain

1994 ◽  
Vol 300 (1) ◽  
pp. 111-115 ◽  
Author(s):  
M H Rider ◽  
K M Crepin ◽  
M De Cloedt ◽  
L Bertrand ◽  
L Hue
Keyword(s):  
Escherichia Coli ◽  
Skeletal Muscle ◽  
Fold Increase ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Wild Type ◽  
Fluorescence Measurements

Asp-130 of the recombinant skeletal-muscle 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase was mutated into Ala in order to study its role in catalysis and/or substrate binding. The D130A mutant displayed a 30- to 140-fold decreased 2-kinase Vmax, depending on the pH, and a 30- and 60-fold increase in Km for MgATP and Fru-6-P respectively at pH 8.5 compared with the wild-type. Mutagenesis of Asp-130 to Ala had no effect on the 2-phosphatase activity, and fluorescence measurements indicated that the changes in kinetic properties of PFK-2 in the D130A mutant were not due to instability. The role of Asp-130 in the 2-kinase reaction is discussed and compared with that of Asp-103 of 6-phosphofructo-1-kinase from Escherichia coli, which binds Mg2+.

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