Direct evidence that Bcr-Abl tyrosine kinase activity disrupts normal synergistic interactions between Kit ligand and cytokines in primary primitive progenitor cells

We previously reported that chronic myelogenous leukemia (CML) primitive granulocyte-monocyte (GM) progenitors have a greatly reduced requirement for kit ligand (KL) to achieve optimal growth with granulocyte colony-stimulating factor (G-CSF) + granulocyte-monocyte colony-stimulating factor (GM-CSF). Conversely, others have demonstrated that unlike normal, CML CD34+ progenitors can proliferate in response to KL as a sole stimulus. To address these seemingly paradoxical findings, we examined the growth responses of CML CD34+ GM progenitors to various cytokines with and without a potent inhibitor of Bcr-Abl tyrosine kinase activity, PD173955. The heightened growth responses of CML GM progenitors to KL alone and to G-CSF + GM-CSF were abrogated by 10 nM PD173955 while having no effect on normal GM progenitors. While normal GM progenitors exhibited the expected synergistic response when KL was added to G-CSF + GM-CSF, CML GM progenitors had a minimal response; however, some synergism was restored by 10 nM PD173955. Normal erythroid progenitors require the synergistic interaction between KL and a saturating amount of erythropoietin (EPO, 1 unit) for optimal growth. In contrast, CML erythroid progenitors had up to 50% of optimal growth in KL alone, and, only a subthreshold amount of EPO (0.1 unit) was needed with KL to achieve 85% of the optimal response; these heightened growth responses were largely abrogated by 10 nM PD173955. Thus, direct evidence is provided that constitutively activated Bcr-Abl kinase pathways in primitive CML progenitors cooperate with single growth factors producing a heightened growth response, and, in so doing, disrupt the normally required synergistic interactions between KL and other cytokines to achieve activation and optimal growth of primitive progenitors. Coupled with our previous findings that a larger than normal proportion of CML primitive progenitors are at a later stage of maturation, we propose that this disruption of normal synergistic responses leads to increased progenitor recruitment into a committed pool by a process of accelerated maturation.     

Ret receptor tyrosine kinase sustains proliferation and tissue maturation in intestinal epithelia

Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. We report a new and physiologically significant site of Ret expression in the intestine: the intestinal epithelium. Experiments in Drosophila indicate that Ret is expressed both by enteric neurons and adult intestinal epithelial progenitors, which require Ret to sustain their proliferation. Mechanistically, Ret is engaged in a positive feedback loop with Wnt/Wingless signalling, modulated by Src and Fak kinases. We find that Ret is also expressed by the developing intestinal epithelium of mice, where its expression is maintained into the adult stage in a subset of enteroendocrine/enterochromaffin cells. Mouse organoid experiments point to an intrinsic role for Ret in promoting epithelial maturation and regulating Wnt signalling. Our findings reveal evolutionary conservation of the positive Ret/Wnt signalling feedback in both developmental and homeostatic contexts. They also suggest an epithelial contribution to Ret loss‐of‐function disorders such as Hirschsprung disease.     

The inhibitory effect of BSP-A1/-A2 on protein kinase C and tyrosine protein kinase

Bovine seminal plasma contains a group of similar proteins, namely BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins), and they are secreted by the seminal vesicles. In our study, we purified the BSP-A1/-A2 through affinity chromatography and found for the first time that BSP-A1/-A2 can inhibit the activity of protein kinase C (PKC) and tyrosine protein kinase (TPK). The inhibition was dose dependent. When the PKC and TPK activities are expressed as the logarithm of percentage activity taking the activity in the absence of the BSP-A1/-A2 as 100%, there is a linear relationship between the their activities and the dose of BSP-A1/-A2.     

Inhibitory effect of piceatannol, a protein tyrosine kinase inhibitor, on asexual maturation of Plasmodium falciparum

This report describes the effect of piceatannol (3,4,3',5'-tetrahydroxy trans stilbene), a plant secondary natural product, on protein tyrosine kinase (PTK) activity in different stages of P. falciparum grown in vitro. Piceatannol inhibited PTK activity in trophozoites and schizonts suggesting that PTK may be important in the initial asexual maturation of the parasite. Inhibition of PTK activity by piceatannol may thus provide new insights into more specific tools for chemotherapeutic interventions for P. falciparum.     

Effect of follicular cells,IGF-I and tyrosine kinase blockers on oocyte maturation

The aim of this study was to test the following hypotheses: (i) that oocyte maturation is controlled by surrounding follicular cells; (ii) that a meiosis-regulating factor of follicular origin is not species-specific; (iii) that one of the follicular regulators of oocyte maturation is IGF-I; and, (iv) that Cumulus oophorus and tyrosine kinase-dependent intracellular mechanisms do not mediate IGF-I action on oocytes. It was found that co-culture of cumulus-enclosed bovine oocytes with isolated bovine ovarian follicles or with isolated porcine ovarian follicles significantly increased the proportion of matured oocytes (at metaphase II of meiosis) after culture. Porcine oocytes without cumulus investments had lower maturation rates than cumulus-enclosed oocytes. Co-culture with isolated porcine ovarian follicles resulted in stimulation of maturation of both cumulus-free and cumulus-enclosed porcine oocytes. These observations suggest that follicular cells (whole follicles or Cumulus oophorus) support bovine and porcine oocyte maturation, and that follicular maturation-promoting factor is not species-specific. The release of significant amounts of IGF-I by cultured bovine and porcine isolated follicles and granulosa cells was demonstrated. Addition of IGF-I to culture medium at 10 or 100 (but not 1000) ng/ml stimulated meiotic maturation of both cumulus-enclosed and cumulus-free porcine oocytes. Neither of the tyrosine kinase blockers, genistein or lavendustin (100 ng/ml medium), changed the stimulating effect of IGF-I on porcine oocytes. The present data suggest that at least one of the follicular stimulators of oocyte nuclear maturation is IGF-I, and that its effect is probably not mediated by cumulus investment or by tyrosine kinase-dependent intracellular mechanisms.     

The effect of angiotensin III on protein tyrosine kinase activity in rat pituitary

Angiotensin III is the biological active peptide from the angiotensin family, which play the important role in several cellular processes. Ang III is the product of reaction catalyzed by aminopeptidase A and in turn can be a substrate for aminopeptidase N, enzyme which converts Ang III to shorter fragment, Ang IV. Aminopeptidase N is specifically inhibited by PC18. Ang III can act by binding to receptors AT1, AT2 or other type of receptors, which are not well recognized. The connection of Ang III to AT1 and AT2 receptors could be inhibited by losartan or PD123319, respectively. The aim of this study was to investigate what is the influence of angiotensin III on protein tyrosine kinase activity in rat pituitary and what is the possible place of interaction of Ang III with target cells. The obtained results show that Ang III can modify tyrosine kinase activity in concentration dependent manner but Ang IV appeared more effective. In presence of PC18 Ang III caused the same changes as Ang III alone that suggests that Ang III, not its metabolite modify tyrosine kinase activity. Losartan and PD123319 given together with Ang III enhanced the changes induced by Ang III. It suggests that the investigated peptide has an effect on protein tyrosine kinase activity in a different way than via AT1 and AT2 receptors.     

The Lyn tyrosine kinase differentially regulates dendritic cell generation and maturation

The Src family kinase Lyn plays both stimulatory and inhibitory roles in hemopoietic cells. In this report we provide evidence that Lyn is involved in dendritic cell (DC) generation and maturation. Loss of Lyn promoted DC expansion in vitro from bone marrow precursors due to enhanced generation and accelerated differentiation of Lyn-deficient DC progenitors. Differentiated Lyn-deficient DCs also had a higher survival rate. Similarly, the CD11c-positive cell number was increased in aged Lyn-deficient mice in vivo. In contrast to their enhanced generation, lyn-/- DCs failed to mature appropriately in response to innate stimuli, resulting in DCs with lower levels of MHC class II and costimulatory molecules. In addition, IL-12 production and Ag-specific T cell activation were reduced in lyn-/- DCs after maturation, resulting in impaired Th1 responses. This is the first study to characterize Lyn-deficient DCs. Our results suggest that Lyn kinase plays uniquely negative and positive regulatory roles in DC generation and maturation, respectively.     

The tec family tyrosine kinase Btk Regulates RANKL-induced osteoclast maturation

A spontaneous mutation in Bruton's tyrosine kinase (Btk) induces a defect in B-cell development that results in the immunodeficiency diseases X-linked agammaglobulinemia in humans and X-linked immunodeficiency (Xid) in mice. Here we show an unexpected role of Btk in osteoclast formation. When bone marrow cells derived from Xid mice were stimulated with receptor activator of NF-kappaB ligand, an osteoclast differentiation factor, they did not completely differentiate into mature multinucleated osteoclasts. Moreover, we found that the defects appeared to occur at the stage in which mononuclear preosteoclasts fuse to generate multinucleated cells. Supporting this notion, macrophages from Xid mice also failed to form multinucleated foreign body giant cells. The fusion defect of the Xid mutant osteoclasts was caused by decreased expression of nuclear factor of activated T cells c1 (NFATc1), a master regulator of osteoclast differentiation, as well as reduced expression of various osteoclast fusion-related molecules, such as the d2 isoform of vacuolar H(+)-ATPase V0 domain and the dendritic cell-specific transmembrane protein. This deficiency was completely rescued by the introduction of a constitutively active form of NFATc1 into bone marrow-derived macrophages. Our data provide strong evidence that Btk plays a critical role in osteoclast multinucleation by modulating the activity of NFATc1.     

Non-circadian rhythm in proliferation of haematopoietic stem cells

The proportion of haematopoietic stem cells (CFU-s) engaged in DNA synthesis was determined by means of the [3H]-thymidine [( 3H]TdR) suicide technique during recovery of bone marrow from the damage caused by a sublethal total body irradiation. In contrast with previous reports the [3H]TdR suicide rate was not permanently increased. It was observed that CFU-s passed through S phase in synchronous waves, following a dose of irradiation of 1.5 Gy. After a dose of 2.6 Gy, there was only one initial wave of increased CFU-s sensitivity to the action of [3H]TdR. Following the depression occurring 26 hr after the irradiation with 2.6 Gy, the proportion of CFU-s killed by the [3H]TdR was permanently increased until 5-6 days after irradiation. Thereafter large differences in the [3H]TdR suicide data were observed among individual mice. Evidence was obtained that individual mice, which had been irradiated by a dose of 2.6 Gy 8-9 days before, had identical values of the CFU-s [3H]TdR suicide rate in the bone marrow from different bones of the lower extremities. The recurrence of the synchronous waves in CFU-s passage through the cell cycle was recorded when the CFU-s population regenerated to only about 10% of its normal value. These waves were obviously not related to a particular time of the day and, consequently, they did not represent the circadian rhythm. It is concluded that the synchronous waves in which CFU-s proliferation occurred reflected the action of the control mechanism on CFU-s proliferation. This mechanism should be endowed with an important systemic component besides locally operating factors.     

Association of inhibitory tyrosine protein kinase p50csk with protein tyrosine phosphatase PEP in T cells and other hemopoietic cells.

p50csk is a tyrosine protein kinase (TPK) that represses the activity of Src family TPKs. We previously showed that Csk is a potent negative regulator of antigen receptor signaling in T lymphocytes and that its Src homology (SH) 3 and SH2 domains are required to inhibit these signals. To test the idea that the Csk SH3 and SH2 domains mediate interactions with other cellular proteins, we attempted to identify Csk-associated polypeptides using the yeast two-hybrid system. The results of our experiments demonstrated that Csk physically associates with PEP, a protein tyrosine phosphatase (PTP) expressed in hemopoietic cells. Further analyses revealed that this interaction was mediated by the Csk SH3 domain and by a proline-rich region (PPPLPERTP) in the non-catalytic C-terminal portion of PEP. The association between Csk and PEP was documented in transiently transfected Cos-1 cells and in a variety of cells of hemopoietic lineages, including T cells. Additional analyses demonstrated that the association between Csk and PEP is highly specific. Together, these data indicated that PEP may be an effector and/or a regulator of p50csk in T cells and other hemopoietic cells. Moreover, they allowed the identification of PEP as the first known ligand for the Csk SH3 domain.     

The effect of choleragen and epidermal growth factor on proliferation and maturation in vitro of human ectocervical cells

Summary The role of choleragen (CT) and epidermal growth factor (EGF) has been examined in relation to the control of growth and differentiation of adult human cervical epithelial (HCE) cells derived from the ectocervix. Cervical biopsies derived from hysterectomy specimens were trypsin disaggregated and HCE cells were plated at 5×10³/cm² in the presence of 2×10⁴/cm² lethally irradiated Swiss 3T3 fibroblasts. Cultures were grown in Liebovitz medium supplemented with 10% fetal bovine serum and hydrocortisone. Epidermal growth factor at 10 ng/ml and choleragen at 10⁻¹⁰ M were added to cultures either singly or in combination. DNA replication in these cultures was measured autoradiographically after exposing cells to tritiated thymidine for 2 h. Differentiation was assessed histochemically by determining glycogen accumulation using the periodic acid Schiff technique. Choleragen increased colony plating efficiency by at least a factor of two but had no effect on colony size Epidermal growth factor did not increase plating efficiency but did increase colony size. In EGF treated colonies DNA replication occurred throughout the colony compared to CT treated colonies in which replication was restricted to the periphery. In the absence of EGF, population doublings achieved in culture did not exceed 32 and glycogen accumulation was evident in cells early in culture life. Colonies treated with EGF exhibited glycogen accumulation late in culture life and the EGF treated cells achieved at least 50 population doublings in culture. The results are discussed in relation to the role of EGF and choleragen on cell differentiation.     

Characterization of protein tyrosine kinase activity in murine Leydig tumor cells

The activity of protein tyrosine kinase (EC 2.7.1.37) was characterized from Leydig tumor cells (M5480A) using the synthetic peptide NH2-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly-COOH as a substrate. Relatively high tyrosine-specific protein kinase activity (about 135 pmol/mg protein per min) was detected in a particulate fraction (30 000 X g pellet) and was found to be linear as a function of time and protein concentration. The enzymic activity in the particulate fraction was stimulated 1.4-fold by 0.02% Nonidet P-40 as judged by 32PO4 incorporated into the peptide. Phosphorylation of endogenous proteins in M5480A particulate fractions with [gamma-32P]ATP resulted in several alkali-resistant radiolabeled bands in polyacrylamide gels in the presence of sodium dodecyl sulfate. Included in this group was a major radiolabeled doublet with an apparent molecular-weight in the range of 50 000-54 000. Phosphoamino acid analysis of hydrolysates of these eluted proteins indicated the presence of phosphotyrosine. Several alkali-resistant radio-labeled bands, including a major doublet with an apparent molecular-weight of 32 000, were also detected after culturing M5480A cells in the presence of 32PO4. These studies demonstrate the presence of high levels of protein tyrosine kinase activity in Leydig tumor cells and of endogenous protein substrates for this enzyme activity.     

Cell cycle distribution of primitive haematopoietic cells stimulated in vitro and in vivo

A novel approach is used to study the proliferating behaviour of primitive haematopoietic cell populations in response to different stimuli. A mathematical model based on the average proportion of apoptotic, dividing and quiescent cells in primitive haematopoietic cell populations is developed to describe the mitotic history of 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester-labelled cells. The cell cycle distributions in different cytokine-supplemented cultures of primitive human and mouse bone marrow cells are determined and compared with those found in vivo. The results indicate that a combination of flt-3 ligand, Steel factor and interleukin-11 or hyper-interleukin-6 provide a level of mitogenic stimulation similar to that existing in vivo after a myeloablative radiation dose. The comparison of the cell cycle distribution obtained for different cultures of human bone marrow CD34(+)(45RA/71)(-) cells demonstrates that the addition of flt-3 ligand in these cultures decreases apoptosis significantly but does not reduce quiescence. In addition, in vivo and in vitro, it was found that more than 3 days of stimulation are required to recruit a maximum number of quiescent cells into active cell cycle. These kinetics of cell cycle activation are found to be similar to those identified for the haematopoietic stem cells compartment in the same cultures. This mathematical analysis provides a useful tool for the development of haematopoietic stem cell culture processes for clinical applications.     

Regulation of the c-Abl and Bcr-Abl tyrosine kinases

The prototypic non-receptor tyrosine kinase c-Abl is implicated in various cellular processes. Its oncogenic counterpart, the Bcr-Abl fusion protein, causes certain human leukaemias. Recent insights into the structure and regulation of the c-Abl and Bcr-Abl tyrosine kinases have changed the way we look at these enzymes.     

LckBP1, a proline-rich protein expressed in haematopoietic lineage cells, directly associates with the SH3 domain of protein tyrosine kinase p56lck.

The Lck tyrosine kinase molecule plays an essential role in T cell activation and T cell development. Using the expression cloning technique, we have isolated a gene that encodes a molecule, LckBP1, able to associate with murine Lck. Analysis of full-length LckBP1 cDNA indicates at least four potentially important segments: a four tandem 37 amino acid repeat motif with a potential helix-turn-helix DNA binding motif; a proline-rich region; a proline-glutamate repeat; and an SH3 domain. These four regions are very similar to the human haematopoietic-specific protein 1 (HS1). Deletion mutant analysis of LckBP1 revealed two proline-rich regions that permit association with Lck SH3. One region contains prolines conserved among HS1 and cortactin, and the other region contains a potential MAP kinase recognition site. In vivo association between Lck and LckBP1 was confirmed by immunoprecipitation of lysates from a pre-T cell line and adult thymocytes using antibodies specific for Lck and LckBP1. LckBP1 is tyrosine phosphorylated after T-cell receptor stimulation. The SH3 domain and the potential helix-turn-helix motif in LckBP1 suggest that this molecule may associate with various molecules and function as a DNA binding molecule. The data also suggest that LckBP1 mediates intracellular signalling through Lck in T cells.     

Characteristics of protein tyrosine kinase activities of Y-79 retinoblastoma cells and retina

Protein tyrosine kinase activities were determined in retina and Y-79 retinoblastoma cells. Kinase activity was associated with particulate subcellular fractions. Specific activities were similar in both retina and Y-79 cells; apparent Km values for ATP and casein were also similar. Retinoblastoma-derived growth factor stimulated endogenous protein tyrosine phosphorylation in Y-79 cells significantly more than in retina. Differences in protein tyrosine phosphorylation between retina and Y-79 retinoblastoma appear to be due to differences in regulation of the activity by the growth factor or differences in the endogenous protein substrates present in the Y-79 cells.