Identification and characterization of a myristylated and palmitylated serine/threonine protein kinase.

We report the molecular cloning and initial characterization of a novel fatty acid acylated serine/threonine protein kinase. The putative open reading frame is predicted to encode a 305 amino acid protein possessing a carboxy-terminal protein kinase domain and amino-terminal myristylation and palmitylation sites. The protein kinase has been accordingly denoted as the myristylated and palmitylated serine/threonine protein kinase (MPSK). Human and mouse MPSKs share approximately 93% identity at the amino acid level with complete retention of acylation sites. Radiation hybridization localized the human MPSK gene to chromosome 2q34-37. Northern analysis demonstrated that the human MPSK 1.7 kilobase mRNA is widely distributed. Epitope tagged human MPSK was found to be acylated by myristic acid at glycine residue 2 and by palmitic acid at cysteines 6 and/or 8. Palmitylation of MPSK in these experiments was found to require an intact myristylation site. While epitope tagged MPSK in immune complexes or purified human glutathione S transferase-MPSK was found to autophosphorylate at one or more threonine residues, the enzyme was not found to phosphorylate several other common exogenous substrates. Indeed, only PHAS-I was identified as an exogenous substrate which was found to be phosphorylated on threonine and serine residues.

Reconstitution of Btk signaling by the atypical tec family tyrosine kinases Bmx and Txk.

Bruton's tyrosine kinase (Btk) is mutated in X-linked agammaglobulinemia patients and plays an essential role in B cell receptor signal transduction. Btk is a member of the Tec family of nonreceptor protein-tyrosine kinases that includes Bmx, Itk, Tec, and Txk. Cell lines deficient for Btk are impaired in phospholipase C-gamma2 (PLCgamma2)-dependent signaling. Itk and Tec have recently been shown to reconstitute PLCgamma2-dependent signaling in Btk-deficient human cells, but it is not known whether the atypical Tec family members, Bmx and Txk, can reconstitute function. Here we reconstitute Btk-deficient DT40 B cells with Bmx and Txk to compare their function with other Tec kinases. We show that in common with Itk and Tec, Bmx reconstituted PLCgamma2-dependent responses including calcium mobilization, extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activation, and apoptosis. Txk also restored PLCgamma2/calcium signaling but, unlike other Tec kinases, functioned in a phosphatidylinositol 3-kinase-independent manner and failed to reconstitute apoptosis. These results are consistent with a common role for Tec kinases as amplifiers of PLCgamma2-dependent signal transduction, but suggest that the pleckstrin homology domain of Tec kinases, absent in Txk, is essential for apoptosis.

Transcriptional analysis of the PTEN/MMAC1 pseudogene, psiPTEN.

PTEN/MMAC1 is a recently characterized tumor suppressor. A pseudogene derived from the human PTEN/MMAC1 phosphatase, psiPTEN, has been reported. Recent analysis of the pseudogene revealed conflicting results about the expression of psiPTEN. In this study, we show that the PTEN/MMAC1 pseudogene is actively transcribed in all cells and tissues examined. In some cases, pseudogene transcripts were found to represent as much as 70% of the total PTEN/MMAC1 RNA. As psiPTEN is transcribed, there is a potential for misinterpretation of PTEN/MMAC1 mutations when RT-PCR techniques are used, as well as potential for a psiPTEN-encoded translation product. Although we were unable to detect a pseudogene protein product in the cell lines examined, a baculovirus produced GST pseudogene fusion protein exhibited phosphatase activity comparable to wild type. The results of this study, taken together, indicate the potential complication of PTEN/MMAC1 molecular analysis caused by the expression of psiPTEN.

Phenotypic analysis of human glioma cells expressing the MMAC1 tumor suppressor phosphatase.

MMAC1, also known as PTEN or TEP-1, was recently identified as a gene commonly mutated in a variety of human neoplasias. Sequence analysis revealed that MMAC1 harbored sequences similar to those found in several protein phosphatases. Subsequent studies demonstrated that MMAC1 possessed in vitro enzymatic activity similar to that exhibited by dual specificity phosphatases. To characterize the potential cellular functions of MMAC1, we expressed wild-type and several mutant variants of MMAC1 in the human glioma cell line, U373, that lacks endogenous expression. While expression of wild-type MMAC1 in these cells significantly reduced their growth rate and saturation density, expression of enzymatically inactive MMAC1 significantly enhanced growth in soft agar. Our observations indicate that while wild-type MMAC1 exhibits activities compatible with its proposed role as a tumor suppressor, cellular expression of MMAC1 containing mutations in the catalytic domain may yield protein products that enhance transformation characteristics.

Selection of murine lymphoid and hematopoietic cells using polystyrene tissue culture devices containing covalently immobilized antibody.

We have established rapid procedures that negatively deplete and positively select for specific murine cell populations. By using polystyrene tissue culture flasks containing a covalently bound mouse anti-rat antibody and specific anti-mouse, cell-surface antigen antibodies, we easily and efficiently depleted greater than 90% of the mature lineage cells from murine bone marrow. This selection procedure resulted in an enrichment of progenitor colonies (CFU-Cs) in murine bone marrow. Using the same polystyrene tissue culture devices, we can directly isolate CD117+ (c-kit+) murine hematopoietic cells. As few as 2000 of these CD117+ cells rescued and reconstituted lethally irradiated recipients in a murine bone marrow transplant model.

Isolation of small, primitive human hematopoietic stem cells: distribution of cell surface cytokine receptors and growth in SCID-Hu mice.

Human CD34+ cells were subfractionated into three size classes using counterflow centrifugal elutriation followed by immunoadsorption to polystyrene cell separation devices. The three CD34+ cell fractions (Fr), Fr 25/29, Fr 33/37, and Fr RO, had mean sizes of 8.5, 9.3 and 13.5 microns, respectively. The majority of cells in the large Fr RO CD34+ cell population expressed the committed stage antigens CD33, CD19, CD38, or HLA-DR and contained the majority of granulocyte-macrophage colony-forming units (CFU-GM), burst-forming units-erythroid (BFU-E), and CFU-mixed lineage (GEMM). In contrast, the small Fr 25/29 CD34+ cells were devoid of committed cell surface antigens and lacked colony-forming activity. When seeded to allogeneic stroma, Fr RO CD34+ cells produced few CFU-GM at week 5, whereas cells from the Fr 25/29 CD34+ cell population showed a 30- to 55-fold expansion of myeloid progenitors at this same time point. Furthermore, CD34+ cells from each size fraction supported ontogeny of T cells in human thymus/liver grafts in severe combined immunodeficient (SCID) mice. Upon cell cycle analyses, greater than 97% of the Fr 25/29 CD34+ cells were in G0/G1 phase, whereas greater proportions of the two larger CD34+ cell fractions were in active cell cycle. Binding of the cytokines interleukin (IL)-1 alpha, IL-3, IL-6, stem cell factor (SCF), macrophage inhibitory protein (MIP)-1 alpha, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage (GM)-CSF to these CD34+ cell populations was also analyzed by flow cytometry. As compared with the larger CD34+ cell fractions, cells in the small Fr 25/29 CD34+ cell population possessed the highest numbers of receptors for SCF, MIP1 alpha, and IL-1 alpha. Collectively, these results indicate that the Fr 25/29 CD34+ cell is a very primitive, quiescent progenitor cell population possessing a high number of receptors for SCF and MIP1 alpha and capable of yielding both myeloid and lymphoid lineages when placed in appropriate in vitro or in vivo culture conditions.

Long-term reconstitution of mice after ex vivo expansion of bone marrow cells: differential activity of cultured bone marrow and enriched stem cell populations.

In this report, we evaluated the short-term expansion of murine bone marrow mononuclear cells (BMMNC) and enriched stem cell populations to determine the capacity of these cells for long-term rescue and engraftment to lethally irradiated recipients. In our study, nonadherent bone marrow mononuclear cell (NBM-MNC) and Thy1+Lin- stem cells populations were cultured with interleukin-3 (IL-3) or IL-3 plus stem cell factor (SCF) for periods up to 6 days. By day 6 of culture, the mononuclear cells (MNC) decreased to 6% of input cell number, whereas Thy1+Lin- cells increased by 2310%. Doses of 95,000; 100,000; 50,000; and 250,000 NBM-MNCs at 0, 1, 2, and 6 days of culture, respectively, rescued 50% of lethally irradiated mice. When 250,000 MNCs were cultured for 0, 1, 2, and 6 days, 71, 61, 100, and 50% of the animals survived lethal irradiation for greater than 24 weeks. In contrast, doses of 8,000 and 21,000 Thy1+Lin- cells cultured 0 and 1 day, respectively, yielded 50% survival rates. These same cells cultured for 6 days failed to rescue recipients even at high doses. Twenty thousand Thy1+Lin- cells cultured for 0, 1, 2, and 6 days, even in the presence of SCF, produced decreasing survival rates of 86, 43, 26, and 0%, respectively. The proliferative responses of these different populations in combination with their long-term rescue abilities indicated that the absolute number of long-term rescue units (LD50, 24 weeks) in the cultured Thy1+Lin- population decreased faster than in similarly cultured NBM-MNCs. Studies evaluating donor cell engraftment demonstrated that animals rescued with cultured Thy1+Lin- and NBM-MNCs maintained high levels of donor reconstitution [7]. The percent donor T cell engraftment did not significantly change between 2 and 17 months post-bone marrow transplantation (post-BMT). Therefore, those animals who received sufficient cells to survive lethal irradiation generally established and maintained high levels of donor engraftment. The data suggest a role for accessory cells and/or factors in the preservation of stem cell activity.

Cloning of a sequence of Aquaspirillum magnetotacticum that complements the aroD gene of Escherichia coli.

A 2 kb DNA fragment isolated from a cosmid library of Aquaspirillum magnetotacticum strain MS-1 complements the aromatic-metabolite requirements and iron-uptake deficiencies of Escherichia coli and Salmonella typhimurium strains that lack a functional aroD (biosynthetic dehydrodquinase) sequence. All recombinant cosmids selected for their aroD complementation property carry this sequence. No DNA sequence homology has, however, been detected by Southern hybridization between the cloned fragment and the aroD gene of E. coli or the qa2 (catabolic dehydroquinase) gene of Neurospora crassa.

Cloning and characterization of the recA gene of Aquaspirillum magnetotacticum.

The recA gene of Aquaspirillum magnetotacticum has been isolated from a genomic library and introduced into a recA mutant strain of Escherichia coli K12. The cloned gene complemented both the recombination and DNA repair deficiency of the host and its protein product promoted the proteolytic cleavage of the LexA protein. A protein whose molecular weight is similar to that of the RecA protein of E. coli was associated with the cloned sequence.