Pim-1 Gene Research Articles

Impaired Interleukin-3 Response in Pim-1-Deficient Bone Marrow-Derived Mast Cells

AbstractThe mouse Pim-1 gene encodes two cytoplasmic serine-threonine—specific protein kinases. The gene has been found to be activated (overexpressed) by retroviral insertion in hematopoietic tumors in mice. Transgenic mice that overexpress Pim-1 (Eμ-Pim-1) have a low incidence of spontaneous T-cell lymphomas and an increased susceptibility to Moloney murine leukemia virus and N-ethyl-N-ni-trosourea-induced lymphomas. Apart from a slight enlargement of the spleen, no abnormalities were found in prelymphomatous transgenic mice. Inactivation of the Pim-1 gene in the germline of mice resulted in mice with a surprisingly subtle phenotype. Therefore, we investigated whether subtle effects of the absence of Pim-1 could be made visible during in vitro culturing of hematopoietic cells. We found that bone marrow—derived mast cells (BMMC) lacking Pim-1 had a distinct growth disadvantage when grown on interleukin (IL)-3, but not when stimulated by the factors IL-4, IL-9, or Steel factor (SF). This indicates a role for Pim-1 as a modulator of the IL-3 signal transduction pathway.

Transcriptional Attenuation of PIM-1 Gene

Levels of the mRNA for PIM-1, a protooncogene encoding a cytoplasmic serine threonine kinase show a wide variation among tissues and cell lines, although this gene is transcribed from a GC- rich housekeeping promoter. Previous studies have failed to identify tissue specific elements in the PIM-I promoter raising the possibility that these elements might reside within the gene. Transient transfections of Luciferase reporter gene constructs into the chronic myelogenous leukemia cell line K562 (which expresses high levels of PIM-1 mRNA) demonstrate that the 1.7kbp PIM-I promoter sequences alone were three times more efficient than constructs driven by the promoter + PIM-1 genomic sequences. Nuclear run on assays of nascent RNA from K562 cells revealed premature transcriptional termination within the PIM-l gene. Thus, PIM-1 gene may be constitutively transcribed in all tissues and transcriptional attenuation could be one of the mechanisms regulating the observed differences in steady state levels of mRNA.

Defects in mRNA 3'-end formation, transcription initiation, and mRNA transport associated with the yeast mutation prp20: possible coupling of mRNA processing and chromatin structure.

A temperature-sensitive lethal mutation in Saccharomyces cerevisiae, prp20-1, causes defects in several different steps in mRNA metabolism, including mRNA 3'-end formation, transcription initiation, and mRNA transport. Previous work has demonstrated that prp20 mutants are defective in actin pre-mRNA splicing. PRP20 is related, both in structure and function, to the RCC1 gene of mammals and the PIM1 gene of Schizosaccharomyces pombe, both of which appear to regulate entry into mitosis and chromosome condensation. In this report we demonstrate that, after a shift of prp20 mutants to the restrictive temperature, transcripts of several genes (CUP1, CYH2, and GAL10) are produced that extend 1-10 kb beyond their normal polyadenylation sites. The failure in 3'-end formation occurs within 1-2 min of the temperature shift. Transcription initiation also is disrupted, in that initiation sites upstream of the normal cap site are used. mRNA transport from nucleus to cytoplasm also is perturbed: In situ hybridization using an oligo(dT) probe demonstrates accumulation of poly(A) in the nucleus, consistent with the accumulation of longer bulk poly(A) (up to approximately 90-100 nucleotides) and with a failure to transport newly synthesized RNA to the cytoplasm. We demonstrate that prp20 and rna1 mutants are very similar, if not identical, with respect to each of these biochemical phenotypes. In light of the putative role of PRP20 in mitotic control, our results suggest a common step in that process and multiple steps in mRNA synthesis and maturation. We speculate that the perturbations in mRNA processing are the result of effects on the chromatin-nascent RNP-transcription complex or misregulation of a cell cycle component that modifies multiple mRNA-processing activities.

Characterization of the testes-specific pim-1 transcript in rat.

The pim-1 proto-oncogene encodes a serine/threonine protein kinase and is expressed in cells of hematolymphoid origin and in the germ cell lineages. In somatic cells, the pim-1 gene is expressed as a 2.8 kb transcript while a shorter sized transcript (2.3 kb) is expressed in rat testes. We have determined that the shorter testes-specific pim-1 transcript arises through the use of an alternate polyadenylation signal present in the 3' untranslated region of the gene. This alternate polyadenylation event results in the removal of an A/U-rich regulatory element located in the 3' untranslated region of the pim-1 gene. This A/U-rich motif has been shown by a number of laboratories to destabilize the transcripts of genes that contain this sequence. Consistent with these findings, we have demonstrated that the shortened testes-specific pim-1 transcript is more stable than the longer A/U-rich containing somatic transcript. We suggest that the functional significance of different sized pim-1 transcripts may be directly related to their different stabilities and that the greater stability of the testes-specific transcript may be essential for the translational delay observed in post-meiotic male germ cells.

Stability changes in pim-1 proto-oncogene mRNA after mitogen stimulation of normal lymphocytes.

mRNA expression of the serine/threonine kinase protooncogene Pim-1 was investigated in mitogen-treated normal bovine lymphocytes. After stimulation with Con A and phorbol ester (PMA), normal bovine PBMC exhibited a 3.5-fold induction of pim-1 mRNA within 4 h of stimulation. By 17 h poststimulation, however, the level of pim-1 mRNA had fallen to approximately 50%. Similar transient kinetics of pim-1 expression were also observed in mitogen-stimulated bovine lymph node lymphocytes and the amount of pim-1 mRNA induced was dependent on the type of mitogen stimulus. Typically, stimulation with Con A and PMA together acted synergistically resulting in a greatly increased amount of pim-1 mRNA induction compared to stimulation with either Con A or PMA alone. To determine if an increase in pim-1 mRNA stability contributed to the overall increase in pim-1 mRNA levels observed after mitogen stimulation, RNA half-life studies were performed. At 4 h poststimulation, pim-1 mRNA in bovine lymph node lymphocytes was relatively stable with a t1/2 of approximately 80 min; at 17 h poststimulation, however, the t1/2 (approximately 35 min) was markedly decreased. In addition, cyclohexamide treatment was found to markedly increase the stability of pim-1 transcripts in bovine PBMC suggesting that a protein synthesis-dependent posttranscriptional pim-1 mRNA degradation pathway may be involved in the regulation of pim-1 mRNA levels in lymphoid cells. To investigate the possible contribution of the destabilizing (UAUU)n motif to pim-1 mRNA stability, the t1/2 of the short 2.4-kb germ cell-specific pim-1 transcript found in rat testes was compared to that of the longer 2.8-kb somatic transcript expressed in stimulated rat lymphocytes. t1/2 determination experiments showed that the 2.4-kb testes-specific transcript, which is missing the destabilizing (UAUU)n sequence, was quite stable (t1/2 much greater than 6 h). In contrast, the t1/2 of the longer 2.8 kb somatic cell pim-1 transcript that contains this A/U-rich sequence motif was found to be much shorter (t1/2 approximately 130 min) in mitogen-activated rat lymphocytes. Together these findings indicate that the transient induction of pim-1 gene expression is associated with normal lymphocyte activation and that the stability of pim-1 transcripts is regulated in lymphocytes during the course of activation. In addition, the differences in pim-1 mRNA stability observed in germ cells and lymphocytes of the same animal are likely the result of molecular mechanisms involving differential regulation by the message destabilizing (UAUU)n motif.

Mapping of the Pim-1 oncogene in mouse t-haplotypes and its use to define the relative map positions of the tcl loci t0( t6) and tw12 and the marker tf (tufted)

Pim-1 is an oncogene activated in mouse T-cell lymphomas induced by Moloney and AKR mink cell focus (MCF) viruses. Pim-1 was previously mapped to chromosome 17 by somatic cell hybrids, and subsequently to the region between the hemoglobin α-chain pseudogene 4 ( Hba-4ps) and the α-crystalline gene ( Crya-1) by Southern blot analysis of DNA obtained from panels of recombinant inbred strains. We have now mapped Pim-1 more accurately in t-haplotypes by analysis of recombinant t-chromosomes. The recombinants were derived from Tt s6tf t 12 parents backcrossed to + tf +tf , and scored for recombination between the loci of T and tf. For simplicity all t-complex lethal genes properly named tck-t x are shortened to t x . The Pim-1 gene was localized 0.6 cM proximal to the t w12 lethal gene, thus placing the Pim-1 gene 5.2 cM distal to the H-2 region in t-haplotypes. Once mapped, the Pim-1 gene was used as a marker for further genetic analysis of t-haplotypes, t w12 is so close to tf that even with a large number of recombinants it was not possible to determine whether it is proximal or distal to tf. Southern blot analysis of DNA from T-tf recombinants with a separation of t w12 and tf indicated that t w12 is proximal to tf. The mapping of two alletic t-lethals, t 0 and t 6 with respect to t w12 and tf has also been a problem. The map position of t 0( t 6) was obtained through Southern blot analysis of DNA from a panel of Tt s6tf t 0 and Tt s6tf t 6 recombinants with separation of t w12 and t 0( t 6). t 0( t 6) was placed distal to tf. Southern blot analysis of the t h20 deletion, which covers the locus of tf, indicated that this deletion does not include the Pim-1 gene.

The pim-1 oncogene encodes two related protein-serine/threonine kinases by alternative initiation at AUG and CUG.

The pim-1 gene is frequently found activated by proviral insertion in murine T cell lymphomas. Overexpression of pim-1 in lymphoid cells by transgenesis formally proved its oncogenic potential. The pim-1 cDNA sequence predicts that both murine and human pim-1 encode a 34 kd protein with homology to protein kinases. In this study, we show that the murine pim-1 gene encodes a 44 kd protein in addition to the predicted 34 kd protein. The 44 kd protein is an amino-terminal extension of the 34 kd protein and is synthesized by alternative translation initiation at an upstream CUG codon. Contrary to previous findings by others, we provide evidence that both murine and human pim-1 gene products are protein-serine/threonine kinases. Murine 44 kd and 34 kd pim-1 proteins exhibit comparable in vitro kinase activity and are both mainly cytoplasmic, but they differ in in vivo association state and half-life.

The Human Pim-1 Gene Is Selectively Transcribed in Different Hemato-Lymphoid Cell Lines in Spite of a G+C-Rich Housekeeping Promoter

The expression of the Pim-1 proto-oncogene was studied by using the K562, Daudi, and Jurkat cell lines. In K562, Pim-1 mRNA levels were more than 20-fold higher than in Daudi and 50-fold higher than in Jurkat. Nuclear run-on assay data correlated directly with the steady-state mRNA levels, suggesting that the rate of transcription was responsible for the selective expression of this gene. Furthermore, the half-life of Pim-1 mRNA was shown to be 47 min in K562, 71 min in Daudi, and 35 min in Jurkat. This indicated that selective Pim-1 mRNA expression did not depend on posttranscriptional regulation. Therefore, 1.7 kilobases of the Pim-1 promoter was sequenced and studied in detail. The sequence showed that the region from nucleotide -1 to -873 was G + C rich (71%). Study of promoter deletions defined two major functional regions, a proximal element (nucleotide -104 to -1) and a distal element (nucleotide -427 to -336). DNase I protection assays identified binding sites for the Sp1 and AP2 proteins in these elements. A possible new transcription factor binds at position -348 in the distal element. In our study of the 1.7-kilobase Pim-1 promoter, we found no differences between K562 and Jurkat that could explain large differences in transcription. Therefore, the Pim-1 promoter appears to function constitutively, and we conclude that distant elements must regulate the tissue-selective expression of this gene. Although the Pim-1 gene has a G + C-rich housekeeping promoter, expression is carefully regulated at the level of transcription.

The human Pim-1 gene is selectively transcribed in different hemato-lymphoid cell lines in spite of a G + C-rich housekeeping promoter.

The expression of the Pim-1 proto-oncogene was studied by using the K562, Daudi, and Jurkat cell lines. In K562, Pim-1 mRNA levels were more than 20-fold higher than in Daudi and 50-fold higher than in Jurkat. Nuclear run-on assay data correlated directly with the steady-state mRNA levels, suggesting that the rate of transcription was responsible for the selective expression of this gene. Furthermore, the half-life of Pim-1 mRNA was shown to be 47 min in K562, 71 min in Daudi, and 35 min in Jurkat. This indicated that selective Pim-1 mRNA expression did not depend on posttranscriptional regulation. Therefore, 1.7 kilobases of the Pim-1 promoter was sequenced and studied in detail. The sequence showed that the region from nucleotide -1 to -873 was G + C rich (71%). Study of promoter deletions defined two major functional regions, a proximal element (nucleotide -104 to -1) and a distal element (nucleotide -427 to -336). DNase I protection assays identified binding sites for the Sp1 and AP2 proteins in these elements. A possible new transcription factor binds at position -348 in the distal element. In our study of the 1.7-kilobase Pim-1 promoter, we found no differences between K562 and Jurkat that could explain large differences in transcription. Therefore, the Pim-1 promoter appears to function constitutively, and we conclude that distant elements must regulate the tissue-selective expression of this gene. Although the Pim-1 gene has a G + C-rich housekeeping promoter, expression is carefully regulated at the level of transcription.

Primary structure of the putative human oncogene, pim-1

A full-length (6.1-kb) human genomic pim-1 gene, together with its immediate 5′-upstream promoter sequence ( Ppim) was isolated and sequenced. The human pim-1 gene shares an overall nucleotide (nt) sequence identity of 53% with the previously reported murine pim-1 gene. It consists of six exons and five introns and contains a protein-coding region that is identical in nt sequence to a full-length human pim-1 cDNA. The gene codes for a predicted Pim-1 protein of 313 amino acids (aa) with an M r of 35 690 and a pI of 5.7. The deduced aa sequence of the human Pim-1 has 94% identity with the murine Pim-1 whereas the nt sequences of the two genes are 88% identical. All of the conserved aa residues of the mouse pim-1 gene, which are homologous to known protein kinases are conserved in the predicted human protein. The human Ppim region is very G + C-rich (69%) and shares greater than 80% identity with the murine Ppim. The Ppim has no TATA- or CAAT-box sequences but does contain a number of nt sequences similar to the putative binding sites of several presumptive transcription factors.

A Pro → Leu substitution in codon 369 of the alpha-1-antitrypsin deficiency variant PI MHeerlen

The molecular defect has been elucidated in the alpha-1-antitrypsin (PI) gene of a patient with a serum level of only 5 mg/100 ml and a PI M-like phenotype, designated PI MHeerlen. The restriction fragment patterns obtained by probes covering the whole gene and flanking sequences were normal, suggesting no major rearrangements. The nucleotide sequence of the exons, intron/exon junctions, and a part of the promoter region is similar to that of a PI M1(Ala213) gene except for an C----T mutation in codon 369, causing a Pro----Leu substitution. Haplotype analysis and oligonucleotide hybridization studies demonstrated the homozygous state of the mutation in the index case. It is most likely that the Pro369----Leu substitution is responsible for the low serum alpha-1-antitrypsin concentration of the patient because this mutation is solely confined to the PI MHeerlen allele and no other relevant mutations could be revealed. As proline is important for the secondary and tertiary structure of proteins, the mutation may cause an abnormal processing of the nascent polypeptide. The same mutation was observed in two unrelated subjects known to carry a PI allele giving a low serum alpha-1-antitrypsin level.

Predisposition to lymphomagenesis in pim-1 transgenic mice: Cooperation with c-myc and N-myc in murine leukemia virus-induced tumors

Transgenic mice bearing the pim-1 gene supplemented with an upstream immunoglobulin enhancer and a downstream murine leukemia virus long terminal repeat express pim-1 mRNA at high levels in both B and T cells. Between 5% and 10% of the pim-1 transgenic mice develop clonal T cell lymphomas before 7 months of age, whereas none of the age-matched control mice do, providing direct evidence for the oncogenic potential of pim-1. Histological examination and FACS analysis revealed no abnormalities in hematopoietic tissues of disease-free pim-1 transgenic mice. When newborn pim-1 transgenic mice are infected with MuLV, T cell lymphomas develop much faster (latency 7-8 weeks) than in nontransgenic mice (latency 22 weeks). In all these T cell lymphomas either c-myc or N-myc was activated by proviral insertion, suggesting strong cooperation between pim-1 and myc in lymphomagenesis.

Identification of the human pim-1 gene product as a 33-kilodalton cytoplasmic protein with tyrosine kinase activity.

The human pim-1 gene was recently identified as a new putative oncogene located on chromosome 6p21, a region showing karyotypic abnormalities in particular leukemias. In the present work we characterized the pim protein product. In vitro translation of positively selected poly(A)+ mRNA indicates that this gene encodes a 33-kilodalton protein. Anti-pim antibodies were raised against a fused TrpE-pim protein induced in a bacterial expression vector. This antibody immunoprecipitated a 33-kilodalton protein from in vivo [35S]methionine-labeled K562 and KCl myelogenous origin cell lines. This protein was localized to the cytoplasm, and in vivo labeling as well as in vitro kinase assay suggests that it is a phosphoprotein with tyrosine kinase activity. This was further confirmed by performing autophosphorylation directly on a p33pim-containing gel band cut out after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results imply that the tyrosine kinase activity of pim can be recovered after boiling the pim-1 protein in sample buffer: a feature not described yet for this class of protein. These results suggest that pim-1 is a new member of the subgroup of oncogenes encoding tyrosine kinases.

Identification of the Human pim-1 Gene Product as a 33-Kilodalton Cytoplasmic Protein with Tyrosine Kinase Activity

The human pim-1 gene was recently identified as a new putative oncogene located on chromosome 6p21, a region showing karyotypic abnormalities in particular leukemias. In the present work we characterized the pim protein product. In vitro translation of positively selected poly(A)+ mRNA indicates that this gene encodes a 33-kilodalton protein. Anti-pim antibodies were raised against a fused TrpE-pim protein induced in a bacterial expression vector. This antibody immunoprecipitated a 33-kilodalton protein from in vivo [35S]methionine-labeled K562 and KCl myelogenous origin cell lines. This protein was localized to the cytoplasm, and in vivo labeling as well as in vitro kinase assay suggests that it is a phosphoprotein with tyrosine kinase activity. This was further confirmed by performing autophosphorylation directly on a p33pim-containing gel band cut out after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results imply that the tyrosine kinase activity of pim can be recovered after boiling the pim-1 protein in sample buffer: a feature not described yet for this class of protein. These results suggest that pim-1 is a new member of the subgroup of oncogenes encoding tyrosine kinases.

Cloning and characterization of the human PIM-1 gene: a putative oncogene related to the protein kinases.

The mouse PIM-1 gene has been implicated in the evolution of retrovirus-associated mouse lymphomas. We have initiated a study of the human PIM-1 gene because of its potential importance as a human oncogene. We have isolated genomic and cDNA clones for this gene and characterized this locus in detail. The predicted PIM-1 protein is 313 amino acids in length. It has homology to a number of the protein kinases but does not have a transmembrane region. The amino acid corresponding to tyrosine-416 of pp60v-src is a tyrosine (position 198), which is consistent with the hypothesis that PIM-1 is a tyrosine kinase rather than a serine-threonine kinase. The PIM-1 gene was found to have six exons and five introns derived from 5 kb of genomic DNA. The site of transcription initiation was localized by S1 nuclease protection studies which indicated that the mature PIM-1 mRNA was approximately 2.7 kb in length. The promotor of this gene had no TATA or CAAT box but did have multiple GC boxes (CCGCCC) that might bind the Sp1 protein. The PIM-1 gene was expressed in myeloid and B lymphoid cell lines, but not in T lymphoid and nonhemopoietic lines. This initial characterization of PIM-1 will allow us to define its role in normal and malignant hematolymphoid differentiation.

Induction of thymomas by N-methyl-N-nitrosourea in AKR mice: interaction between the chemical carcinogen and endogenous murine leukaemia viruses.

AKR mice develop thymomas spontaneously when greater than 6 months old but when young AKR mice are treated with N-methyl-N-nitrosourea (MNU) they develop thymomas at 3-6 months of age. In this study the potential role of oncogene activation in the development of both the spontaneous and MNU-induced thymomas in AKR mice has been examined by DNA transfection into NIH3T3 mouse fibroblasts and by Southern analysis of tumour DNA. The results show that a high proportion of MNU-induced thymomas contain activated cellular rasK while no activated cellular ras genes were detected in spontaneous thymomas. Southern analysis of tumour DNA revealed that 2/30 spontaneous tumours and 2/52 MNU-induced tumours contained alterations in the c-myc gene while 5/29 spontaneous tumours and 6/56 MNU-induced tumours contained alterations in the Pim-1 gene. A more detailed analysis of the Pim-1 gene demonstrated that the alterations observed in most MNU-induced and spontaneous tumours resulted from proviral integration at the 3' end of this gene. Our analyses also demonstrated that the majority of MNU-induced tumours, including those containing rearrangements in the Pim-1 gene, lacked the somatically acquired recombinant MCF proviruses that are present in most spontaneous AKR lymphomas. These results provide evidence that the mechanisms of development of MNU-induced and spontaneous tumours in AKR mice are distinct and the development of thymomas that contain proviral integrations at the Pim-1 locus in the MNU-treated AKR mice involve cooperation between the chemical carcinogen and endogenous murine leukaemia viruses.

The cDNA sequence and gene analysis of the human pim oncogene

The putative oncogene pim-1 is frequently activated by provirus insertion in MuLV-induced T cell lymphomas in mice. By analogy with other cellular oncogenes that are similarly activated (e.g., myc, myb, erbB) it is possible that pim-1 may also be involved in some human tumors. To study human pim-1 we cloned and sequenced human pim-1 cDNA clones. The human pim-1 codes for a protein of 313 amino acids (aa) which is highly homologous (94%) to the deduced amino acid sequence of mouse pim-1. All the mouse pim-1 residues which are homologous to protein kinases are conserved in the human pim-1. We also isolated the human pim-1 gene and mapped it relative to the cDNA. The RNA transcript of human pim-1 is approx. 3.0 kb and it is highly expressed in the erythroleukemia cell line K562.

The primary structure of the putative oncogene pim-1 shows extensive homology with protein kinases

We have shown previously that the putative oncogene pim-1 is frequently activated by provirus insertion in murine leukemia virus-induced T cell lymphomas. Here we describe the structure of the pim-1 gene as determined by sequencing genomic and cDNA clones. The gene has an open reading frame, encoding a protein of 313 amino acids, extending over six exons and preceded and followed by stop codons in all reading frames. Proviruses always integrate outside the protein-encoding domain, showing a high preference for a small region in the 3′-terminal exon; integration in the 3′ exon results in relatively high levels of pim-1 mRNA. Computer search reveals homology between pim-1 and protein kinases: all the domains characteristic of protein kinases are conserved in the pim-1 amino acid sequence. The highest homologies were observed with the protein-serine kinases.

Localization of the human pim oncogene (PIM) to a region of chromosome 6 involved in translocations in acute leukemias.

The human homolog, hpim, of the murine pim-1 gene, which is activated in murine T-cell lymphomas by insertion of retrovirus proviral genomes in the pim-1 region, has been molecularly cloned; the cloned probe has been used to map the hpim locus to human chromosome region 6p21 by somatic cell hybrid analysis and chromosomal in situ hybridization. The hpim gene is expressed as a 3.2-kilobase mRNA in various human cell lines of hematopoietic lineage, most dramatically in the K562 erythroleukemia cell line, which contains a cytogenetically demonstrable rearrangement in the 6p21 region. A characteristic chromosome anomaly, a reciprocal translocation t(6;9)(p21;q33), has been described in myeloid leukemias and could involve the hpim gene.

Genetic mapping of Pim-1 putative oncogene to mouse chromosome 17.

Pim-1 is a putative oncogene activated in T-cell lymphomas induced by Moloney and AKR mink cell focus forming (MCF) viruses. We have determined the chromosomal localization of the Pim-1 gene in mice by Southern blot analysis of DNAs obtained from a panel of mouse-Chinese hamster somatic cell hybrids. The Pim-1 gene was localized on chromosome 17, a chromosome frequently aberrant in T-cell lymphomas. Two chromosomal regions, containing sequences homologous to regions within the Pim-1 locus, were localized on chromosome 6 and 16.