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Biomedical subjects

O N Witte

Publications and source records attributed to O N Witte.

At least 91 records · Page 5Linked to original sources
PubMed1995

Tyrosine phosphorylation of BCR by FPS/FES protein-tyrosine kinases induces association of BCR with GRB-2/SOS.

The human bcr gene encodes a protein with serine/threonine kinase activity, CDC24/dbl homology, a GAP domain, and an SH2-binding region. However, the precise physiological functions of BCR are unknown. Coexpression of BCR with the cytoplasmic protein-tyrosine kinase encoded by the c-fes proto-oncogene in Sf-9 cells resulted in stable BCR-FES protein complex formation and tyrosine phosphorylation of BCR. Association involves the SH2 domain of FES and a novel binding domain localized to the first 347 amino acids of the FES N-terminal region. Deletion of the homologous N-terminal BCR-binding domain from v-fps, a fes-related transforming oncogene, abolished transforming activity and tyrosine phosphorylation of BCR in vivo. Tyrosine phosphorylation of BCR in v-fps-transformed cells induced its association with GRB-2/SOS, the RAS guanine nucleotide exchange factor complex. These data provide evidence that BCR couples the cytoplasmic protein-tyrosine kinase and RAS signaling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Isolation of unknown genes from human bone marrow by differential screening and single-pass cDNA sequence determination.

A cDNA sequencing project was initiated to characterize gene expression in human bone marrow and develop strategies to isolate novel genes. Forty-eight random DNAs from total human bone marrow were subjected to single-pass DNA sequence analysis to determine a limited complexity of mRNAs expressed in the bone marrow. Overall, 8 cDNAs (17%) showed no similarity to known sequences. Information from DNA sequence analysis was used to develop a differential prescreen to subtract unwanted cDNAs and to enrich for unknown cDNAs. Forty-eight cDNAs that were negative with a complex probe were subject to single-pass DNA sequence determination. Of these prescreened cDNAs, the number of unknown sequences increased to 23 (48%). Unknown cDNAs were also characterized by RNA expression analysis using 25 different human leukemic cell lines. Of 13 unknown cDNAs tested, 10 were expressed in all cell types tested and 3 revealed a hematopoietic lineage-restricted expression pattern. Interestingly, while a total of only 96 bone marrow cDNAs were sequenced, 31 of these cDNAs represent sequences from unknown genes and 12 showed significant similarities to sequences in the data bases. One cDNA revealed a significant similarity to a serine/threonine-protein kinase at the amino acid level (56% identity for 123 amino acids) and may represent a previously unknown kinase. Differential screening techniques coupled with single-pass cDNA sequence analysis may prove to be a powerful and simple technique to examine developmental gene expression.

Amino Acid Sequence↗
PubMed1994

Binding of beta gamma subunits of heterotrimeric G proteins to the PH domain of Bruton tyrosine kinase.

Bruton tyrosine kinase (Btk) has been implicated as the defective gene in both human and murine B-cell deficiencies. The identification of molecules that interact with Btk may shed light on critical processes in lymphocyte development. The N-terminal unique region of Btk contains a pleckstrin homology domain. This domain is found in a broad array of signaling molecules and implicated to function in protein-protein interactions. By using an in vitro binding assay and an in vivo competition assay, the pleckstrin homology domain of Btk was shown to interact with the beta gamma dimer of heterotrimeric guanine nucleotide-binding proteins (G proteins). A highly conserved tryptophan residue in subdomain 6 of the pleckstrin homology domain was shown to play a critical role in the binding. The interaction of Btk with beta gamma suggests the existence of a unique connection between cytoplasmic tyrosine kinases and G proteins in cellular signal transduction.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1994

Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-cell antigen receptor surface engagement.

We evaluated in WEHI 231 B cells the time-dependent responses of Lyn, Blk, Btk, Syk, and three members of the Jak family of protein tyrosine kinases following antibody-mediated surface engagement of the B-cell antigen receptor. Our results show that the enzyme activities of Lyn and Blk were stimulated within seconds of antigen receptor engagement and correlated with the initial tyrosine phosphorylation of the Ig alpha and Ig beta subunits of the B-cell antigen receptor. Btk enzyme activity was also transiently stimulated and was maximal at approximately 5 min after B-cell receptor surface binding. Syk activity gradually increased to a maximum at 10-30 min following receptor ligation and was found to parallel the association of Syk with the tyrosine phosphorylated Ig alpha and Ig beta subunits of the receptor. While the specific activities of the Jak1, Jak2, and Tyk2 protein tyrosine kinases were unaltered following B-cell receptor ligation, the abundance of Jak1 and Jak2 were increased 3- to 4-fold within 10 min of receptor engagement. These results demonstrate that multiple families of non-transmembrane protein tyrosine kinases are temporally regulated during the process of B-cell antigen receptor-initiated intracellular signal transduction.

Amino Acid Sequence↗
PubMed1994

Deletion within the Src homology domain 3 of Bruton's tyrosine kinase resulting in X-linked agammaglobulinemia (XLA).

The gene responsible for X-linked agammaglobulinemia (XLA) has been recently identified to code for a cytoplasmic tyrosine kinase (Bruton's agammaglobulinemia tyrosine kinase, BTK), required for normal B cell development. BTK, like many other cytoplasmic tyrosine kinases, contains Src homology domains (SH2 and SH3), and catalytic kinase domain. SH3 domains are important for the targeting of signaling molecules to specific subcellular locations. We have identified a family with XLA whose affected members have a point mutation (g-->a) at the 5' splice site of intron 8, resulting in the skipping of coding exon 8 and loss of 21 amino acids forming the COOH-terminal portion of the BTK SH3 domain. The study of three generations within this kinship, using restriction fragment length polymorphism and DNA analysis, allowed identification of the mutant X chromosome responsible for XLA and the carrier status in this family. BTK mRNA was present in normal amounts in Epstein-Barr virus-induced B lymphoblastoid cell lines established from affected family members. Although the SH3 deletion did not alter BTK protein stability and kinase activity of the truncated BTK protein was normal, the affected patients nevertheless have a severe B cell defect characteristic for XLA. The mutant protein was modeled using the normal BTK SH3 domain. The deletion results in loss of two COOH-terminal beta strands containing several residues critical for the formation of the putative SH3 ligand-binding pocket. We predict that, as a result, one or more crucial SH3 binding proteins fail to interact with BTK, interrupting the cytoplasmic signal transduction process required for B cell differentiation.

Adult↗
PubMed1994

Differential complementation of Bcr-Abl point mutants with c-Myc.

A complementation strategy was developed to define the signaling pathways activated by the Bcr-Abl tyrosine kinase. Transformation inactive point mutants of Bcr-Abl were tested for complementation with c-Myc. Single point mutations in the Src-homology 2 (SH2) domain, the major tyrosine autophosphorylation site of the kinase domain, and the Grb-2 binding site in the Bcr region impaired the transformation of fibroblasts by Bcr-Abl. Hyperexpression of c-Myc efficiently restored transformation activity only to the Bcr-Abl SH2 mutant. These data support a model in which Bcr-Abl activates at least two independent pathways for transformation. This strategy may be useful for discerning signaling pathways activated by other oncogenes.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Role of Bruton's tyrosine kinase in immunodeficiency.

The genetic defect associated with human X-linked agammaglobulinemia and murine X-linked immunodeficiency was recently shown to result from lack of function of a new cytoplasmic tyrosine kinase, called Bruton's tyrosine kinase (Btk). The phenotypes associated with these immunodeficiencies indicate that Btk plays a critical role in B-lymphocyte development. The distinctive protein structure of Btk and preliminary functional studies suggest that Btk may act in a novel manner in a variety of signaling pathways.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1994

Functional analysis of the AUG- and CUG-initiated forms of the c-Myc protein.

Activation of the c-myc proto-oncogene by chromosomal translocation or proviral insertion frequently results in the separation of the c-myc coding region from its normal regulatory elements. Such rearrangements are often accompanied by loss or mutation of c-myc exon 1 sequences. These genetic alterations do not affect synthesis of the major c-myc protein, p64, which is initiated from the first AUG codon in exon 2. However they can result in mutation or loss of the CUG codon located in exon 1 that normally serves as an alternative translational initiation codon for synthesis of an N-terminally extended form of c-Myc (p67). It has been hypothesized that p67 is a functionally distinct form of c-Myc whose specific loss during c-myc rearrangements confers a selective growth advantage. Here we describe experiments designed to test the functional properties of the two c-Myc protein forms. We introduced mutations within the translational initiation codons of a normal human c-myc cDNA that alter the pattern of Myc protein synthesis (p64 vs. p67). The functions of each of these proteins were experimentally addressed using co-transformation and transcriptional activation assays. Both the p64 and p67 c-Myc proteins were independently able to collaborate with bcr-abl in the transformation of Rat-1 fibroblasts. In addition, both the exon 1- and exon 2-initiated forms of the c-Myc protein stimulated transcription of a Myc/Max-responsive reporter construct to a similar level. Given the apparent absence of functional differences between p64 and p67, we conclude that the basis for c-Myc oncogenic activation lies primarily in the overall deregulation of its expression and not in alterations in the protein. The existence of the CUG translational initiator may reflect a mechanism for the continued synthesis of c-Myc protein under conditions where AUG initiation is inhibited.

Amino Acid Sequence↗
PubMed1994

Tyrosine phosphorylation and activation of Bruton tyrosine kinase upon Fc epsilon RI cross-linking.

Tyrosine phosphorylation of several cellular proteins is one of the earliest signaling events induced by cross-linking of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on mast cells or basophils. Tyrosine kinases activated during this process include the Src family kinases, Lyn, c-Yes, and c-Src, and members of another subfamily, Syk and PTK72 (identical or highly related to Syk). Recently, some of us described two novel tyrosine kinases, Emb and Emt, whose expression was limited to subsets of hematopoietic cells, including mast cells. Emb turned out to be identical to Btk, a gene product defective in human X-linked agammaglobulinemia and in X-linked immunodeficient (xid) mice. Here we report that Fc epsilon RI cross-linking induced rapid phosphorylation on tyrosine, serine, and threonine residues and activation of Btk in mouse bone marrow-derived mast cells. A small fraction of Btk translocated from the cytosol to the membrane compartment following receptor cross-linking. Tyrosine phosphorylation of Btk was not induced by either a Ca2+ ionophore (A23187), phorbol 12-myristate 13-acetate, or a combination of the two reagents. Co-immunoprecipitation between Btk and receptor subunit beta or gamma was not detected. The data collectively suggest that Btk is not associated with Fc epsilon but that its activation takes place prior to protein kinase C activation and plays a novel role in the Fc epsilon RI signaling pathway.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1993

En bloc substitution of the Src homology region 2 domain activates the transforming potential of the c-Abl protein tyrosine kinase.

Src homology region 2 (SH2) domains are present in many proteins involved in signal transduction. In nonreceptor protein tyrosine kinases the SH2 domain has been implicated in regulation of tyrosine kinase activity and in mediating interactions involved in downstream signaling. Different SH2 domains exhibit distinct binding specificities for both phosphotyrosine- and phosphoserine/phosphothreonine-containing proteins. We show that different SH2 domains are not functionally equivalent within the context of the c-ABL1b protooncogene. c-ABL1b, altered by replacement of its SH2 domain with the N-terminal SH2 domain of Ras GTPase-activating protein, exhibited activated transforming capability, caused intracellular tyrosine phosphorylation of p62, and was relocalized from nucleus to cytoplasm. This en bloc substitution apparently uncouples two distinct functions of the SH2 domain so that c-ABL escapes normal regulatory control while it retains the capability to elicit signals that promote transformation. The SH2 domain of the ARG protein tyrosine kinase, which shares high amino acid-sequence homology with the SH2 domain of ABL, was less effective in activating the oncogenic potential of c-ABL. The effects that the N-terminal SH2 domain of Ras GTPase-activating protein has in the context of c-ABL resemble the effects of deleting the SH3 domain. Thus, the SH2 and SH3 domains may have coordinate roles as regulatory control elements within the context of c-ABL.

Animals↗
PubMed1993

Efficient transplantation of BCR-ABL-induced chronic myelogenous leukemia-like syndrome in mice.

Lethally irradiated mice reconstituted with bone marrow expressing P210 BCR-ABL can develop myeloproliferative syndromes that resemble the initial phase of human chronic myelogenous leukemia (CML). Mice that develop the CML-like syndrome can be segregated into two groups based on the latency with which the granulocytic disease appears--early onset (< 20 weeks) and late onset (> 20 weeks). Only cells from mice exhibiting the late-onset CML-like syndrome can efficiently propagate the disease when transplanted into sublethally irradiated syngeneic recipients. Mice engrafted with late-onset murine CML cells develop a range of hematopoietic disorders that originate from multipotent stem cells. The chronic granulocytic hyperplasia can be propagated by serial transplantation into secondary and tertiary recipient mice. The majority of transplanted mice succumb to acute myeloid and B- and T-lymphoid leukemias. These data support the idea that late-onset murine CML originates from a multipotent progenitor cell with a high replicating capacity. The inability to transplant the disease from mice developing the early-onset CML-like syndrome suggests that this disorder may originate from more differentiated progenitor cells with limited replication capacity that have undergone clonal expansion but are not immortalized. Although both early- and late-onset CML-like syndromes exhibit granulocytic hyperplasia, these disorders represent distinct diseases that appear to originate from different hematopoietic cell types. The late-onset CML-like disease and transfer to secondary recipients provides a useful murine model with features of the chronic and acute phases of human CML.

Animals↗
PubMed1993

Distinctive growth requirements and gene expression patterns distinguish progenitor B cells from pre-B cells.

Long-term bone marrow cultures have been useful in determining gene expression patterns in pre-B cells and in the identification of cytokines such as interleukin 7 (IL-7). We have developed a culture system to selectively grow populations of B lineage restricted progenitors (pro-B cells) from murine bone marrow. Pro-B cells do not grow in response to IL-7, Steel locus factor (SLF), or a combination of the two. c-kit, the SLF receptor, and the IL-7 receptor are both expressed by pro-B cells, indicating that the lack of response is not simply due to the absence of receptors. Furthermore, SLF is not necessary for the growth of pro-B cells since they could be expanded on a stromal line derived from Steel mice that produces no SLF. IL-7 responsiveness in pre-B cells is associated with an increase in n-myc expression and is correlated with immunoglobulin (Ig) gene rearrangements. Although members of the ets family of transcription factors and the Pim-1 kinase are expressed by pro-B cells, n-myc is not expressed. Pro-B cells maintain Ig genes in the germline configuration, which is correlated with a low level of recombination activating genes 1 and 2 (Rag-1 and 2) mRNA expression, but high expression of sterile mu and terminal deoxynucleotidyl transferase. Pro-B cells are unable to grow separated from the stromal layer by a porous membrane, indicating that stromal contact is required for growth. These results suggest that pro-B cells are dependent on alternative growth signals derived from bone marrow stroma and can be distinguished from pre-B cells by specific patterns of gene expression.

Animals↗
PubMed1993

BCR-ABL and v-abl oncogenes induce distinct patterns of thymic lymphoma involving different lymphocyte subsets.

The human BCR-ABL oncogenes encoded by the Philadelphia chromosome (Ph) affect the pathogenesis of diverse types of leukemia and yet are rarely associated with T-lymphoid leukemia. To determine whether BCR-ABL kinases are inefficient in transforming T lymphocytes, BCR-ABL-expressing retroviruses were injected intrathymically into mice. Thymomas that expressed BCR-ABL kinase developed after a relatively long latent period. In most thymomas, deletion of 3' proviral sequences resulted in loss of tk-neo and occasionally caused expression of kinase-active carboxy-terminally truncated BCR-ABL oncoprotein. In contrast, deletion of 3' proviral sequences was not observed in thymomas induced with Abelson murine leukemia virus (A-MuLV). BCR-ABL viruses induced distinct patterns of disease and involved different thymocyte subsets than A-MuLV and Moloney murine leukemia virus (Mo-MuLV). While Mo-MuLV only induced Thy-1+ thymomas, v-abl- and BCR-ABL-induced thymomas often contained mixed populations of B220+ and Thy-1+ lymphocytes in the same tumor. In most v-abl and BCR-ABL tumors, Thy-1+ lymphoid cells expressed CD8 and a continuum of CD4 ranging from negative to positive. Conversely, Mo-MuLV thymomas contained distinct populations of CD4+ cells that were either CD8+ or CD8-. A-MuLV-transformed T-lymphoid cells did not express the CD3/T-cell receptor complex, while BCR-ABL tumors were CD3+. Thus, BCR-ABL viruses preferentially induce somewhat more differentiated T lymphocytes than are transformed by A-MuLV. Furthermore, rare B220+ lymphocytes may represent preferred v-abl and BCR-ABL transformation targets in the thymus.

Animals↗