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 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.

Structural differences between repressed and derepressed forms of p60c-src

1989 ◽  
Vol 9 (6) ◽  
pp. 2648-2656
Author(s):  
A MacAuley ◽  
J A Cooper
Keyword(s):  
Kinase Activity ◽  
Kinase Domain ◽  
Terminal Region ◽  
Phosphorylation Sites ◽  
Carboxy Terminus ◽  
Kinase Activation ◽  
Amino Terminal ◽  
Structural Differences ◽  
Carboxy Terminal ◽  
Pronase E

The kinase activity of p60c-src is derepressed by removal of phosphate from Tyr-527, mutation of this residue to Phe, or binding of a carboxy-terminal antibody. We have compared the structures of repressed and active p60c-src, using proteases. All forms of p60c-src are susceptible to proteolysis at the boundary between the amino-terminal region and the kinase domain, but there are several sites elsewhere that are more sensitive to trypsin digestion in repressed than in derepressed forms of p60c-src. The carboxy-terminal tail (containing Tyr-527) is more sensitive to digestion by pronase E and thermolysin when Tyr-527 is not phosphorylated. The kinase domain fragment released with trypsin has kinase activity. Relative to intact p60c-src, the kinase domain fragment shows altered substrate specificity, diminished regulation by the phosphorylated carboxy terminus, and novel phosphorylation sites. The results identify parts of p60c-src that change conformation upon kinase activation and suggest functions for the amino-terminal region.

Evolution, expression, and chromosomal location of a novel receptor tyrosine kinase gene, eph

1988 ◽  
Vol 8 (9) ◽  
pp. 3770-3776
Author(s):  
Y Maru ◽  
H Hirai ◽  
M C Yoshida ◽  
F Takaku
Keyword(s):  
Tyrosine Kinase ◽  
Blot Analysis ◽  
Protein Tyrosine Kinase ◽  
Normal Liver ◽  
Cell Types ◽  
Kinase Domain ◽  
Kinase Gene ◽  
Preferential Expression ◽  
Cdna Sequences ◽  
Carboxy Terminal

Partial sequence analysis of the genomic eph locus revealed that the splicing points of kinase domain-encoding exons were completely distinct from those of the other protein tyrosine kinase members reported, suggesting that this is the earliest evolutionary split within this family. In Northern (RNA) blot analysis, the eph gene was expressed in liver, lung, kidney, and testis of rat, and screening of 25 human cancers of various cell types showed preferential expression in cells of epithelial origin. Overexpression of eph mRNA was found in a hepatoma and a lung cancer without gene amplification. Comparison of cDNA sequences derived from a normal liver and a hepatoma that overproduces eph mRNA demonstrated that two of them were completely identical throughout the transmembrane to the carboxy-terminal portions. Southern blot analysis of DNAs from human-mouse hybrid clones with an eph probe showed that this gene was present on human chromosome 7.

scholarly journals The Hox cooperativity motif of the chimeric oncoprotein E2a-Pbx1 is necessary and sufficient for oncogenesis.

1997 ◽  
Vol 17 (1) ◽  
pp. 81-88 ◽  
Author(s):  
C P Chang ◽  
I de Vivo ◽  
M L Cleary
Keyword(s):  
Dna Binding ◽  
Single Point ◽  
Point Mutations ◽  
Cell Types ◽  
Site Directed Mutagenesis ◽  
Binding Assays ◽  
Cellular Assays ◽  
Potential Contact ◽  
Carboxy Terminal ◽  
Necessary And Sufficient

E2a-Pbx1 chimeric oncoproteins result from fusion of the E2A and PBX1 genes at the sites of t(1;19) chromosomal translocations in a subset acute lymphoblastic leukemias. Experimentally, E2a-Pbx1 transforms a variety of cell types, including fibroblasts, myeloid progenitors, and lymphoblasts. Structure-function studies have shown that contributions from both E2a and Pbx1 are necessary for oncogenesis, but the Pbx1 homeodomain is dispensable and the required portion of Pbx1 has not been delineated. In this study, we used deletional and site-directed mutagenesis to identify portions of Pbx1 necessary for oncogenic and transcriptional activities of E2a-Pbx1. These studies defined a motif (named the Hox cooperativity motif [HCM]) carboxy terminal to the Pbx homeodomain that is required for cooperative DNA binding, cellular transcriptional activity, and the oncogenic potential of E2a-Pbx1. The HCM is highly conserved throughout the Pbx/exd subfamily of divergent homeodomain proteins and functions in DNA-binding assays as a potential contact site for Hox dimerization. E2a-Pbx1 proteins with interstitial deletion or single-point mutations in the HCM could neither activate transcription in cellular assays nor transform NIH 3T3 cells. An E2a-Pbx1 mutant containing 50 amino acids of Pbx1b spanning the HCM but lacking the homeodomain was capable of inducing fibroblast transformation. Thus, the HCM is a necessary and sufficient contribution of Pbx1 for oncogenesis induced by E2a-Pbx1 and accounts for its homeodomain-independent transforming properties. Since subtle alterations of the Pbx HCM result in complete abrogation of transforming activity whereas the homeodomain is entirely dispensable, we conclude that interactions mediated by the HCM are more important for transformation by E2a-Pbx1 than interactions with cognate Pbx DNA sites.

Leukemic Stem Cells of Chronic Phase CML Patients Possess Uniquely Elevated BCR-ABL Kinase Activity and Acquire Spontaneous BCR-ABL Kinase Domain Mutations at a High Frequency.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 438-438 ◽  
Author(s):  
Xiaoyan Jiang ◽  
Kyi Min Saw ◽  
Allen Eaves ◽  
Connie Eaves
Keyword(s):  
Stem Cells ◽  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Point Mutations ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Tyrosine Kinase Activity ◽  
Abl Kinase ◽  
Kinase Domain Mutations

Abstract Growing evidence indicates that the therapeutic potential of imatinib mesylate (IM) for the treatment of CML may be limited initially by a relative innate resistance of the leukemic stem cells and eventually by an accumulation of cells with BCR-ABL tyrosine kinase domain mutations. We now show that the amount and tyrosine kinase activity of p210-BCR-ABL in the most primitive and relatively IM-unresponsive lin−CD34+CD38− CML cells is 3 to 10-fold higher than in the majority of the lin−CD34+CD38+ CML progenitors (n=3). These results confirm previous BCR-ABL transcript data and identify elevated p210-BCR-ABL expression to be a likely important factor in the characteristic IM-insensitivity of very primitive CML cells. To determine whether in vivo, CML stem cells also accumulate gene mutations affecting the BCR-ABL kinase domain, cDNAs were prepared from RNA extracts of purified lin−CD34+CD38− cells isolated from 3 chronic phase patients that had not received IM therapy. Bidirectional sequencing of individually cloned cDNAs from these samples revealed BCR-ABL kinase domain mutations in 2 of the 3 patients at frequencies of 10% (1/10), 20% (2*/10,*identical mutations). Incubation of these lin−CD34+CD38− cells in vitro for 2–3 wk ± a high concentration of IM (up to 10 μM, which was sufficient to reduce the tyrosine kinase activity in the input cells by 70±12% and in their 2 wk progeny by 10±5%) selected a subpopulation of more differentiated and completely IM-resistant cells. This was shown in Western blots by the inability of 10 μM IM to reduce either their p210-BCR-ABL tyrosine kinase activity or CrkL phosphorylation and in methylcellulose assays ±5 μM IM. As predicted, IM-selected cells showed a higher frequency of kinase domain mutations (13–20% vs 0–20% of cDNA clones analyzed from 3 wk cells cultured ±IM). Analysis of individual colonies produced from CFCs in the cultured cells showed all (21/21) colonies from IM-selected cells had mutations vs 50% (5/10) in those cultured without IM. The total frequency of mutant cDNAs detected was also increased in the IM-resistant cells (35–55% vs 10–25% mutant cDNAs in selected vs control cells). Interestingly, in most cases, both wild-type and mutant cDNAs were identified in the same colony, indicating de novo generation of mutations in vitro. Overall, >50 different mutations were identified. These included 10 point mutations previously associated with clinical IM resistance (including G250 and T315), another 13 point mutations previously identified in a comprehensive mutational screen, and >20 previously undescribed mutations. Several of the latter affect the critical region of the P loop, the c-helix and the activation loop and would be predicted to confer significant IM resistance. To investigate the possibility that the observed genomic instability of very primitive CML cells might be related to their elevated innate p210-BCR-ABL activity, BCR-ABL transcript levels in individual IM-selected, fully resistant and control (similarly treated but no IM exposure) colonies were compared. This showed that BCR-ABL transcripts were ~20-fold higher (P<0.05) in the resistant colonies (30 assessed from 3 patients). These findings suggest that the increased BCR-ABL expression and activity that uniquely characterizes the most primitive CML cells may contribute not only to their innate insensitivity to IM but also to a deregulation of genomic stability leading to the emergence of IM-resistant mutants and other subclones associated with disease progression.

Enhanced SH3:Linker Interaction Suppresses Activating Mutations of the c-Abl Protein-Tyrosine Kinase.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1208-1208
Author(s):  
Shoghag Panjarian ◽  
Shugui Chen ◽  
John Engen ◽  
Thomas Smithgall
Keyword(s):  
Tyrosine Kinase ◽  
Myristic Acid ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Binding Pocket ◽  
Sh3 Domain ◽  
Kinase Domain ◽  
Abl Kinase ◽  
Defective Mutant ◽  
T315i Mutation

Abstract Abstract 1208 Bcr-Abl, the chimeric protein-tyrosine kinase expressed as a result of the Philadelphia chromosome translocation, plays a pivotal role in the initiation and maintenance of chronic myelogenous leukemia (CML). Imatinib (Gleevec) is an ATP-competitive Bcr-Abl inhibitor that selectively kills Bcr-Abl+ CML cells. Despite its clinical success, imatinib is less effective in the advanced stages of CML due to the emergence of drug resistance caused by point mutations in the Abl kinase domain. Second generation Bcr-Abl inhibitors such as dasatinib and nilotinib are active against most imatinib-resistant forms of Bcr-Abl, with the exception of the T315I “gatekeeper” mutant. The Abl gatekeeper residue (Thr315) is located between the ATP-binding site and an adjacent hydrophobic pocket, and forms a key hydrogen bond with imatinib. Additionally, the T315I mutation produces a strong activating effect on the downregulated c-Abl “core,” consisting of the myristoylated N-terminal Ncap, tandem SH3 and SH2 regulatory domains, the SH2-kinase linker, which forms a polyproline type II helix for internal SH3 docking, and the tyrosine kinase domain. Using hydrogen-exchange mass spectrometry, we recently found that the T315I mutation not only induced conformational changes in the Abl kinase domain as expected, but also at a distance in the RT-loop of the SH3 domain. Such changes may allosterically contribute to kinase domain activation by disturbing the negative regulatory influence of SH3:linker interaction. Recently, a new class of allosteric Bcr-Abl inhibitors has been reported that targets the myristate-binding pocket of Abl, which localizes to C-lobe of the kinase domain and away from the active site. Together with our finding that the T315I mutation perturbs SH3:linker interaction, these inhibitors support the existence of an extensive network of allosteric interactions that work together to regulate Abl kinase activity. In this project, we investigated whether enhanced SH3:linker interaction can allosterically reverse the activating effects of the T315I imatinib resistance mutation as well as mutations of the N-terminal myristoylation site and myristic acid binding pocket. We created modified versions of Abl [High Affinity Linker proteins (HALs)] by mutating multiple residues within the SH2-kinase linker to proline, thereby enhancing the SH3 domain binding affinity. Using mammalian cell-based expression assays and immunoblotting with phosphospecific antibodies, we identified five of eleven Abl-HAL proteins that did not exhibit changes in basal kinase activity. The Abl-HAL protein with the greatest enhancement of SH3:linker interaction was then combined with the T315I mutation, a myristoylation-defective mutant, and a myristic acid binding pocket mutation. Remarkably, this HAL substitution completely reversed the activating effect of the myristic acid binding pocket mutation, while substantially suppressing the activity of Abl T315I and the myristoylation-defective mutant. These results indicate that stabilization of SH3:linker interaction allosterically represses Abl activation by a wide variety of mechanisms, and suggests a new approach to allosteric control of Bcr-Abl kinase activity. Disclosures: No relevant conflicts of interest to declare.

Identification of BCR-ABL point mutations conferring resistance to the Abl kinase inhibitor AMN107 (nilotinib) by a random mutagenesis study

Blood ◽  
2007 ◽  
Vol 109 (11) ◽  
pp. 5011-5015 ◽  
Author(s):  
Arghya Ray ◽  
Sandra W. Cowan-Jacob ◽  
Paul W. Manley ◽  
Jürgen Mestan ◽  
James D. Griffin
Keyword(s):  
Imatinib Mesylate ◽  
Kinase Inhibitor ◽  
Single Point ◽  
Point Mutations ◽  
Random Mutagenesis ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Multiple Point ◽  
Resistance Mutations ◽  
Mutagenesis Study

Abstract Patients with advanced stages of chronic myeloid leukemia (CML) often manifest imatinib mesylate resistance associated with point mutations in BCR-ABL. AMN107 is a new higher-potency inhibitor of BCR-ABL. To identify mutations in BCR-ABL that could result in resistance to AMN107, a cDNA library of BCR-ABL mutants was introduced into Ba/F3 cells followed by selection in AMN107 (0.125-0.5 μM). A total of 86 individual, drug-resistant colonies were recovered, and the SH3, SH2, and kinase domains of BCR-ABL were sequenced. A total of 46 colonies had single point mutations in BCR-ABL, with a total of 17 different mutations, all within the kinase domain. The other 40 colonies had multiple point mutations and were not analyzed further. Each of the 17 single point mutants were reconstructed by site-directed mutagenesis of native BCR-ABL and found to be approximately 2.5- to 800-fold more resistant to AMN107 than native BCR-ABL. The mutations included 6 known imatinib mesylate–resistant mutations, including T315I, which showed complete resistance to AMN107. Interestingly, most AMN107-resistant mutants were also resistant to imatinib mesylate. These results may predict some of the resistance mutations that will be detected in clinical trials with this kinase inhibitor.

scholarly journals Modulation of erbB kinase activity and oncogenic potential by single point mutations in the glycine loop of the catalytic domain.

1994 ◽  
Vol 14 (10) ◽  
pp. 6868-6878 ◽  
Author(s):  
H K Shu ◽  
C M Chang ◽  
L Ravi ◽  
L Ling ◽  
C M Castellano ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Conformational Changes ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Single Point ◽  
Point Mutations ◽  
Full Length ◽  
Site Directed Mutagenesis ◽  
Tyrosine Kinase Domain ◽  
Amino Terminal

Avian c-erbB is activated to a leukemia oncogene following truncation of its amino-terminal ligand-binding domain by retroviral insertion. The insertionally activated transcripts encode protein products which have constitutive tyrosine kinase activity and can induce erythroleukemia but not sarcomas. We have previously found that a valine-to-isoleucine point mutation at position 157 (V157I mutant) within the tyrosine kinase domain of this truncated erbB can dramatically activate the sarcomagenic potential of the oncogene and increase the kinase activity of this oncoprotein. This mutation lies at position 157 of the insertionally activated c-erbB product, affecting a highly conserved valine residue of the glycine loop involved in ATP binding and phosphate transfer. To investigate the functional importance of this residue in the catalytic activity of kinases, we have introduced at this position, by site-directed mutagenesis, codons representing the remaining 18 amino acid residues. Most of the mutants have diminished activity, with six of them completely devoid of kinase activity, indicating the sensitivity of this region to conformational changes. Some of these mutants displayed increased kinase activity and greater transforming potential in comparison with IA c-erbB, but none had levels as high as those of the V157I mutant. In general, the sarcomagenic potential of the various erbB mutants correlated with their autophosphorylation state and their ability to cause phosphorylation of MAP kinase. However, there are important exceptions such as the V157G mutant, which lacks enhanced autophosphorylation but is highly sarcomagenic. Studies of this and other autophosphorylation site mutants point to the existence of an autophosphorylation-independent pathway in sarcomagenesis. The requirement for leukemogenic potential is much less stringent and correlates with positivity of kinase activity. When the valine-to-isoleucine substitution was put in context of the full-length erbB protein, the mutation relaxed the ligand dependence and had a positive effect on the transforming potential of the full-length c-erbB.

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 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.

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