Two point mutations in the transmembrane domain of P68gag-ros inactive its transforming activity and cause a delay in membrane association.

None of the 28 identified point mutations in tafazzin (Taz1p), which is the mutant gene product associated with Barth syndrome (BTHS), has a biochemical explanation. In this study, endogenous Taz1p was localized to mitochondria in association with both the inner and outer mitochondrial membranes facing the intermembrane space (IMS). Unexpectedly, Taz1p does not contain transmembrane (TM) segments. Instead, Taz1p membrane association involves a segment that integrates into, but not through, the membrane bilayer. Residues 215–232, which were predicted to be a TM domain, were identified as the interfacial membrane anchor by modeling four distinct BTHS mutations that occur at conserved residues within this segment. Each Taz1p mutant exhibits altered membrane association and is nonfunctional. However, the basis for Taz1p dysfunction falls into the following two categories: (1) mistargeting to the mitochondrial matrix or (2) correct localization associated with aberrant complex assembly. Thus, BTHS can be caused by mutations that alter Taz1p sorting and assembly within the mitochondrion, indicating that the lipid target of Taz1p is resident to IMS-facing leaflets.

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The myristylation signal of p60v-src functionally complements the N-terminal fps-specific region of P130gag-fps

Molecular and Cellular Biology ◽

10.1128/mcb.9.5.2214-2219.1989 ◽

1989 ◽

Vol 9 (5) ◽

pp. 2214-2219

Author(s):

A R Brooks-Wilson ◽

E Ball ◽

T Pawson

Keyword(s):

Tyrosine Kinases ◽

Protein Tyrosine Kinase ◽

Specific Region ◽

Protein Tyrosine Kinases ◽

Membrane Association ◽

Modular Construction ◽

Specific Domain ◽

Protein Tyrosine ◽

Sarcoma Virus ◽

Transforming Activity

The P130gag-fps protein-tyrosine kinase of Fujinami sarcoma virus contains an N-terminal fps-specific domain (Nfps) that is important for oncogenicity. The N-terminal 14 amino acids of p60v-src, which direct myristylation and membrane association, can replace the gag-Nfps sequences of P130gag-fps (residues 1 to 635), producing a highly transforming src-fps polypeptide. Conversely, gag-Nfps can restore modest transforming activity to a nonmyristylated v-src polypeptide. These results emphasize the modular construction of protein-tyrosine kinases and indicate that Nfps, possibly in conjunction with gag, functions in the subcellular localization of P130gag-fps.

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Only the Substitution of Methionine 918 with a Threonine and Not with Other Residues Activates RET Transforming Potential*

Endocrinology ◽

10.1210/endo.138.4.5073 ◽

1997 ◽

Vol 138 (4) ◽

pp. 1450-1455 ◽

Cited By ~ 2

Author(s):

Anna Maria Cirafici ◽

Giuliana Salvatore ◽

Gabriella De Vita ◽

Francesca Carlomagno ◽

Nina A. Dathan ◽

...

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Multiple Endocrine Neoplasia ◽

Multiple Endocrine Neoplasia Type ◽

Point Mutations ◽

Early Response ◽

Specific Point ◽

Medullary Thyroid ◽

Transforming Activity ◽

Early Response Genes

Abstract Specific point-mutations of the RET receptor tyrosine kinase protooncogene are responsible for the inheritance of multiple endocrine neoplasia type 2A (MEN2A) and 2B (MEN2B), and familial medullary thyroid carcinoma (FMTC). MEN2B is caused by the substitution of methionine 918 by a threonine in the tyrosine kinase (TK) domain of RET. This mutation converts RET into a dominant transforming oncogene. We have substituted Met918 with four different residues and found that RET acquired transforming activity only when Met918 was substituted with a threonine. However, also when serine and valine, but not leucine or phenylalanine, were inserted in position 918, the RET TK function was activated and induced, especially in the case of the RET(918Ser), immmediate-early response genes. We conclude that the preservation of Met918 is critical for the control of RET kinase. However, only when a threonine residue is present in position 918, does RET efficiently couple with a transforming pathway.

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The first seven amino acids encoded by the v-src oncogene act as a myristylation signal: lysine 7 is a critical determinant.

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2435 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2435-2441 ◽

Cited By ~ 59

Author(s):

J M Kaplan ◽

G Mardon ◽

J M Bishop ◽

H E Varmus

Keyword(s):

Amino Acids ◽

Rous Sarcoma Virus ◽

Subcellular Location ◽

Membrane Association ◽

Recognition Sequence ◽

Critical Determinant ◽

Amide Linkage ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

Transforming Activity

The transforming protein of Rous sarcoma virus, pp60v-src, is covalently coupled to myristic acid by an amide linkage to glycine 2. Myristylation promotes the association of pp60v-src with cellular membranes, and this subcellular location is essential for transforming activity. The findings presented here, in conjunction with the previous reports of others, imply that the seventh amino acid encoded by v-src might be important in the myristylation reaction. Replacement of lysine 7 by asparagine greatly reduced the myristylation, membrane association, and transforming activity of pp60v-src. In contrast, substitution of arginine at residue 7 had no effect on any of these properties of pp60v-src. Addition of amino acids 1 to 7 encoded by v-src was sufficient to cause myristylation of a src-pyruvate kinase fusion protein. We conclude that the recognition sequence for myristylation of pp60v-src comprises amino acids 1 to 7 and that lysine 7 is a critical component of this sequence.

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Saturation Mutagenesis of the β Subunit of the Human Granulocyte-Macrophage Colony-Stimulating Factor Receptor Shows Clustering of Constitutive Mutations, Activation of ERK MAP Kinase and STAT Pathways, and Differential β Subunit Tyrosine Phosphorylation

Blood ◽

10.1182/blood.v92.6.1989 ◽

1998 ◽

Vol 92 (6) ◽

pp. 1989-2002 ◽

Cited By ~ 22

Author(s):

Brendan J. Jenkins ◽

Timothy J. Blake ◽

Thomas J. Gonda

Keyword(s):

Map Kinase ◽

Tyrosine Phosphorylation ◽

Transmembrane Domain ◽

Point Mutations ◽

Saturation Mutagenesis ◽

Β Subunit ◽

Colony Stimulating Factor ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

Abstract The high-affinity receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are heterodimeric complexes consisting of cytokine-specific  subunits and a common signal-transducing β subunit (hβc). We have previously demonstrated the oncogenic potential of this group of receptors by identifying constitutively activating point mutations in the extracellular and transmembrane domains of hβc. We report here a comprehensive screen of the entire hβc molecule that has led to the identification of additional constitutive point mutations by virtue of their ability to confer factor independence on murine FDC-P1 cells. These mutations were clustered exclusively in a central region of hβc that encompasses the extracellular membrane-proximal domain, transmembrane domain, and membrane-proximal region of the cytoplasmic domain. Interestingly, most hβc mutants exhibited cell type-specific constitutive activity, with only two transmembrane domain mutants able to confer factor independence on both murine FDC-P1 and BAF-B03 cells. Examination of the biochemical properties of these mutants in FDC-P1 cells indicated that MAP kinase (ERK1/2), STAT, and JAK2 signaling molecules were constitutively activated. In contrast, only some of the mutant β subunits were constitutively tyrosine phosphorylated. Taken together, these results highlight key regions involved in hβc activation, dissociate hβc tyrosine phosphorylation from MAP kinase and STAT activation, and suggest the involvement of distinct mechanisms by which proliferative signals can be generated by hβc. © 1998 by The American Society of Hematology.

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Activation of Ha-ras p21 by substitution, deletion, and insertion mutations.

Molecular and Cellular Biology ◽

10.1128/mcb.5.8.1809 ◽

1985 ◽

Vol 5 (8) ◽

pp. 1809-1813 ◽

Cited By ~ 19

Author(s):

R G Chipperfield ◽

S S Jones ◽

K M Lo ◽

R A Weinberg

Keyword(s):

Amino Acid ◽

Point Mutations ◽

Normal Function ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Transforming Activity ◽

Ras P21 ◽

Ras Oncogenes ◽

Insertion Mutations

The transforming activity of naturally arising ras oncogenes results from point mutations that affect residue 12 or 61 of the encoded 21-kilodalton protein (p21). By use of site-directed mutagenesis, we showed that deletions and insertions of amino acid residues in the region of residue 12 are also effective in conferring oncogenic activity on p21. Common to these various alterations is the disruption that they create in this domain of the protein, which we propose results in the inactivation of a normal function of the protein.

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Identification of CD20 Mutations in Malignant Lymphoma: Can They Be Predictors of Response to Rituximab?.

Blood ◽

10.1182/blood.v106.11.288.288 ◽

2005 ◽

Vol 106 (11) ◽

pp. 288-288 ◽

Cited By ~ 3

Author(s):

Yasuhito Terui ◽

Takuma Sakurai ◽

Yuji Mishima ◽

Yuko Mishima ◽

Natsuhiko Sugimura ◽

...

Keyword(s):

Flow Cytometry ◽

Malignant Lymphoma ◽

Laser Scanning ◽

Transmembrane Domain ◽

Point Mutations ◽

K562 Cells ◽

Cytoplasmic Domain ◽

Isolated Cells ◽

Mammalian Expression ◽

Cd20 Expression

Abstract Purpose: Although rituximab is commonly used as induction and maintenance therapy for CD20+ malignant lymphoma, some patients become refractory to treatment and the mechanism of resistance is unclear. The aim of this study was to investigate the relationship between CD20 mutations and rituximab resistance. Methods: To investigate whether CD20 mutations affect the response to rituximab, fresh CD19+ lymphoma cells were isolated from the lymph nodes, peripheral blood or bone marrow of 48 patients with NHL using magnetic activated cell sorting (MACS). CD19+/CD20+ cells were subsequently sorted by flow cytometry. RNA was prepared from the isolated cells and RT-PCR was performed. The resulting PCR products were sequenced, subcloned into the mammalian expression vector pTARGET, transfected into K562 cells and CD20 expression was examined by flow cytometry and laser scanning confocal microscopy. Results: In all 48 patients, overall response rate (CR+CRu+PR) to rituximab was 93.8% (45/48), but two cases became PD after PR. DNA sequence analysis revealed that point mutations were mostly observed in two CD20 domains - the third transmembrane domain and the C-terminal cytoplasmic domain. One patient had point mutations in the transmembrane domain, three cases showed point mutations in the C-terminal cytoplasmic domain and six cases had non-specific CD20 mutations, which did not affect CD20 expression. Thirty-eight patients showed no mutations of CD20 gene. CD20 expression was very weak in patients with point mutations in the C-terminal cytoplasmic domain, whereas expression was increased in patients with point mutations in the transmembrane domain. Conclusions: This study suggests that point mutations in CD20 may cause rituximab resistance and identification of CD20 mutations upon diagnosis may help to predict a patient’s response to rituximab.

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Point mutation of C-terminal region of CD20 molecule predicts rituximab-induced complement-dependent cytotoxicity and clinical response to rituximab in non-Hodgkin’s lymphoma

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.7563 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 7563-7563 ◽

Cited By ~ 2

Author(s):

Y. Terui ◽

Y. Mishima ◽

M. Yokoyama ◽

K. Hatake ◽

...

Keyword(s):

Flow Cytometry ◽

Laser Scanning ◽

Transmembrane Domain ◽

Point Mutations ◽

K562 Cells ◽

Cytoplasmic Domain ◽

Laser Scanning Confocal Microscopy ◽

Cytoplasmic Domains ◽

Isolated Cells ◽

Cd20 Expression

7563 Background: Although rituximab is commonly used as induction and maintenance therapy for malignant lymphoma, some patients become refractory to treatment and the mechanism of resistance is unclear. The aim of this study was to investigate the relationship between CD20 mutations and rituximab resistance. Methods: To investigate whether CD20 mutations affect response to rituximab, fresh CD19+ lymphoma cells were isolated from the lymph nodes, or bone marrow of 68 patients with NHL. The cells were subsequently sorted by flow cytometry. RNA was prepared from the isolated cells and RT-PCR was performed. The PCR products were sequenced, subcloned into an expression vector pTARGET, transfected into K562 cells. CD20 expression was examined by flow cytometry and laser scanning confocal microscopy. Results: In all 68 patients, overall response rate (CR+CRu+PR) to rituximab was 91.2% (62/68), but t four cases became PD after PR. DNA sequence analysis revealed that point mutations were mostly observed in three CD20 domains - extracellular/cytoplasmic domains, the third transmembrane domain and the C-terminal cytoplasmic domain. Two cases had point mutations in extracellular/cytoplasmic domains, one patient had point mutations in the transmembrane domain, four cases showed point mutations in the C-terminal cytoplasmic domain and six cases had non-specific CD20 mutations, which did not affect CD20 expression. 56 patients showed no mutations of CD20 gene. CD20 expression was very weak in patients with point mutations in the C-terminal cytoplasmic domain, whereas expression was increased in patients with point mutations in the transmembrane domain. Conclusions: Point mutations in CD20 may cause rituximab resistance and identification of CD20 mutations upon diagnosis may help to predict a patient’s response to rituximab. No significant financial relationships to disclose.

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Novel imatinib-sensitive PDGFRA-activating point mutations in hypereosinophilic syndrome induce growth factor independence and leukemia-like disease

Blood ◽

10.1182/blood-2010-05-286757 ◽

2011 ◽

Vol 117 (10) ◽

pp. 2935-2943 ◽

Cited By ~ 57

Author(s):

Christian Elling ◽

Philipp Erben ◽

Christoph Walz ◽

Marie Frickenhaus ◽

Mirle Schemionek ◽

...

Keyword(s):

Growth Factor ◽

Hypereosinophilic Syndrome ◽

Point Mutations ◽

Imatinib Treatment ◽

Presumptive Diagnosis ◽

Activating Mutations ◽

Transforming Activity ◽

Selection For ◽

Constitutive Phosphorylation

Abstract The FIP1L1-PDGFRA fusion is seen in a fraction of cases with a presumptive diagnosis of hypereosinophilic syndrome (HES). However, because most HES patients lack FIP1L1-PDGFRA, we studied whether they harbor activating mutations of the PDGFRA gene. Sequencing of 87 FIP1L1-PDGFRA–negative HES patients revealed several novel PDGFRA point mutations (R481G, L507P, I562M, H570R, H650Q, N659S, L705P, R748G, and Y849S). When cloned into 32D cells, N659S and Y849S and—on selection for high expressors—also H650Q and R748G mutants induced growth factor–independent proliferation, clonogenic growth, and constitutive phosphorylation of PDGFRA and Stat5. Imatinib antagonized Stat5 phosphorylation. Mutations involving positions 659 and 849 had been shown previously to possess transforming potential in gastrointestinal stromal tumors. Because H650Q and R748G mutants possessed only weak transforming activity, we injected 32D cells harboring these mutants or FIP1L1-PDGFRA into mice and found that they induced a leukemia-like disease. Oral imatinib treatment significantly decreased leukemic growth in vivo and prolonged survival. In conclusion, our data provide evidence that imatinib-sensitive PDGFRA point mutations play an important role in the pathogenesis of HES and we propose that more research should be performed to further define the frequency and treatment response of PDGFRA mutations in FIP1L1-PDGFRA–negative HES patients.

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Multiple Sequence Elements Facilitate Chp Rho GTPase Subcellular Location, Membrane Association, and Transforming Activity

Molecular Biology of the Cell ◽

10.1091/mbc.e05-09-0896 ◽

2006 ◽

Vol 17 (7) ◽

pp. 3108-3121 ◽

Cited By ~ 28

Author(s):

Emily J. Chenette ◽

Natalia Y. Mitin ◽

Channing J. Der

Keyword(s):

Amino Acids ◽

Rho Gtpases ◽

Rho Gtpase ◽

Subcellular Location ◽

Small Gtpases ◽

Membrane Association ◽

Basic Amino Acids ◽

Sequence Elements ◽

Transforming Activity ◽

Carboxy Terminal

Cdc42 homologous protein (Chp) is a member of the Rho family of small GTPases and shares significant sequence and functional similarity with Cdc42. However, unlike classical Rho GTPases, we recently found that Chp depends on palmitoylation, rather than prenylation, for association with cellular membranes. Because palmitoylation alone is typically not sufficient to promote membrane association, we evaluated the possibility that other carboxy-terminal residues facilitate Chp subcellular association with membranes. We found that Chp membrane association and transforming activity was dependent on the integrity of a stretch of basic amino acids in the carboxy terminus of Chp and that the basic amino acids were not simply part of a palmitoyl acyltransferase recognition motif. We also determined that the 11 carboxy-terminal residues alone were sufficient to promote Chp plasma and endomembrane association. Interestingly, stimulation with tumor necrosis factor-α activated only endomembrane-associated Chp. Finally, we found that Chp membrane association was not disrupted by Rho guanine nucleotide dissociation inhibitory proteins, which are negative regulators of Cdc42 membrane association and biological activity. In summary, the unique carboxy-terminal sequence elements that promote Chp subcellular location and function expand the complexity of mechanisms by which the cellular functions of Rho GTPases are regulated.

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