Ubiquitin-mediated interaction of p210 BCR/ABL with ß-catenin supports disease progression in a murine model for chronic myelogenous leukemia.

We have identified a ubiquitin-binding domain within the NH2-terminal sequences of p210 BCR/ABL and determined that the binding site co-localizes with the binding site for ß-catenin. The domain does not support the auto- or trans-kinase activity of p210 BCR/ABL or its ability to interact with GRB2 and activate ERK1/2 signaling. Expression of p210 BCR/ABL, but not a ß-catenin-binding mutant, in hematopoietic cells is associated with the accumulation of p-ß-catenin (Tyr654) and increased TCF/LEF-mediated transcription. In a bone marrow transplantation model, the interaction between ß-catenin and p-ß-catenin (Tyr654) is detectable in mice transplanted with p210 BCR/ABL, but not the mutant. Whereas mice transplanted with p210 BCR/ABL exhibit myeloid disease with expansion of monocytes and neutrophils, mice transplanted with the mutant predominantly exhibit expansion of neutrophils, polycythemia, and increased lifespan. The increased disease latency is associated with expansion of megakaryocyte-erythrocyte progenitors, a decrease in common myeloid progenitors, and reduced ß-catenin signaling in the bone marrow of the diseased mice. These observations support a model in which p210 BCR/ABL may influence lineage-specific leukemic expansion by directly binding and phosphorylating ß-catenin and altering its transcriptional activity. They further suggest that the interaction may play a role in chronic phase disease progression.

The evolution and expression of the snaR family of small non-coding RNAs.

We recently identified the snaR family of small non-coding RNAs that associate in vivo with the nuclear factor 90 (NF90/ILF3) protein. The major human species, snaR-A, is an RNA polymerase III transcript with restricted tissue distribution and orthologs in chimpanzee but not rhesus macaque or mouse. We report their expression in human tissues and their evolution in primates. snaR genes are exclusively in African Great Apes and some are unique to humans. Two novel families of snaR-related genetic elements were found in primates: CAS (catarrhine ancestor of snaR), limited to Old World Monkeys and apes; and ASR (Alu/snaR-related), present in all monkeys and apes. ASR and CAS appear to have spread by retrotransposition, whereas most snaR genes have spread by segmental duplication. snaR-A and snaR-G2 are differentially expressed in discrete regions of the human brain and other tissues, notably including testis. snaR-A is up-regulated in transformed and immortalized human cells, and is stably bound to ribosomes in HeLa cells. We infer that snaR evolved from the left monomer of the primate-specific Alu SINE family via ASR and CAS in conjunction with major primate speciation events, and suggest that snaRs participate in tissue- and species-specific regulation of cell growth and translation.

Bcr interacts with components of the endosomal sorting complex required for transport-I and is required for epidermal growth factor receptor turnover.

Virtually all patients with chronic myelogenous leukemia (CML) express an aberrant protein (p210 Bcr-Abl) that contains NH2-terminal sequences from Bcr fused to COOH-terminal sequences from Abl. In a yeast two-hybrid screen, we have identified TSG101 as a binding partner for Bcr. Because TSG101 is a subunit of the mammalian endosomal sorting complex required for transport (ESCRT), which regulates protein sorting during endosomal trafficking, this association suggests that Bcr may have a related cellular function. The docking site for TSG101 has been mapped to the COOH terminus of Bcr, indicating that this interaction may be disrupted in CML. Overexpression studies with full-length TSG101 and Bcr reveal that this interaction can be recapitulated in mammalian cells. The association can also be observed between natively expressed proteins in a panel of hematopoietic and nonhematopoietic cell lines, where a second subunit of the ESCRT complex, vacuolar sorting protein 28 (Vps28), was also found to interact with Bcr. Both Bcr and TSG101 exhibit a punctate cytoplasmic distribution and seem to colocalize in HeLa cells, which would be consistent with an in vivo association. Bacterially purified Bcr and TSG101 also bind, suggesting that the interaction is direct and is not dependent on ubiquitination. Disruption of the endosomal pathway with an ATPase-defective Vps4 mutant results in the cellular redistribution of Bcr, and suppression of Bcr in HeLa cells by small interfering RNA impairs epidermal growth factor receptor turnover. Taken together, these observations suggest that Bcr is a component of the mammalian ESCRT complexes and plays an important role in cellular trafficking of growth factor receptors.

Induction of extracellular matrix-remodeling genes by the senescence-associated protein APA-1.

Human fibroblasts undergo cellular senescence after a finite number of divisions, in response to the erosion of telomeres. In addition to being terminally arrested in the cell cycle, senescent fibroblasts express genes that are normally induced upon wounding, including genes that remodel the extracellular matrix. We have identified the novel zinc finger protein APA-1, whose expression increased in senescent human fibroblasts independent of telomere shortening. Extended passage, telomerase-immortalized fibroblasts had increased levels of APA-1 as well as the cyclin-dependent kinase inhibitor p16. In fibroblasts, APA-1 was modified by the ubiquitin-like protein SUMO-1, which increased APA-1 half-life, possibly by blocking ubiquitin-mediated degradation. Overexpression of APA-1 did not cause cell cycle arrest; but, it induced transcription of the extracellular matrix-remodeling genes MMP1 and PAI2, which are associated with fibroblast senescence. MMP1 and PAI2 transcript levels also increased in telomerase-immortalized fibroblasts that had high levels of APA-1, demonstrating that the matrix-remodeling phenotype of senescent fibroblasts was not induced by telomere attrition alone. APA-1 was able to transactivate and bind to the MMP1 promoter, suggesting that APA-1 is a transcription factor that regulates expression of matrix-remodeling genes during fibroblast senescence.

Regulation of the multifunctional Ca2+/calmodulin-dependent protein kinase II by the PP2C phosphatase PPM1F in fibroblasts.

The regulation of the multifunctional calcium/calmodulin dependent protein kinase II (CaMKII) by serine/threonine protein phosphatases has been extensively studied in neuronal cells; however, this regulation has not been investigated previously in fibroblasts. We cloned a cDNA from SV40-transformed human fibroblasts that shares 80% homology to a rat calcium/calmodulin-dependent protein kinase phosphatase that encodes a PPM1F protein. By using extracts from transfected cells, PPM1F, but not a mutant (R326A) in the conserved catalytic domain, was found to dephosphorylate in vitro a peptide corresponding to the auto-inhibitory region of CaMKII. Further analyses demonstrated that PPM1F specifically dephosphorylates the phospho-Thr-286 in autophosphorylated CaMKII substrate and thus deactivates the CaMKII in vitro. Coimmunoprecipitation of CaMKII with PPM1F indicates that the two proteins can interact intracellularly. Binding of PPM1F to CaMKII involves multiple regions and is not dependent on intact phosphatase activity. Furthermore, overexpression of PPM1F in fibroblasts caused a reduction in the CaMKII-specific phosphorylation of the known substrate vimentin(Ser-82) following induction of the endogenous CaM kinase. These results identify PPM1F as a CaM kinase phosphatase within fibroblasts, although it may have additional functions intracellularly since it has been presented elsewhere as POPX2 and hFEM-2. We conclude that PPM1F, possibly together with the other previously described protein phosphatases PP1 and PP2A, can regulate the activity of CaMKII. Moreover, because PPM1F dephosphorylates the critical autophosphorylation site of CaMKII, we propose that this phosphatase plays a key role in the regulation of the kinase intracellularly.

Termination of lifespan of SV40-transformed human fibroblasts in crisis is due to apoptosis.

Normal human fibroblasts in culture have a limited lifespan, ending in replicative senescence. Introduction of SV40 sequences encoding large T antigen and small t antigen into pre-senescent cells results in an extension of lifespan for an additional 20-30 population doublings. Rare clones of SV40-transformed cells are capable of indefinite growth and are described as immortal; however, the great majority of SV40-transformed cells terminate this extended lifespan in cell death, termed "crisis." We have examined the properties of cells in crisis to obtain further insights into mechanism of cell death and immortalization. Populations at the terminal cell passage show a balance between cell replication and cell death over a period of several weeks, with a progressive increase in cells undergoing cell death. During this period, there is less than a 3-fold increase in attached cell number, with two stages being identifiable on the basis of the focal pattern of cell survival. We also demonstrate that cells in crisis are undergoing apoptosis based on TUNEL assay, subG1 DNA content, annexin V reactivity, and activation of caspases 3 and 8. We suggest a model whereby SV40-transformed cells acquire increased sensitivity to apoptosis based on changes in properties which activate caspase 8 in addition to changes previously described involving shortening of telomeric sequences. While only telomere stabilization could be clearly shown to be essential for survival of cells through crisis, the extended period of cell replication and altered gene expression observed in SV40-transformed cells during crisis are compatible with other genetic alterations in immortal cells.