Regulation of protein phosphotyrosine content by changes in tyrosine kinase and protein phosphotyrosine phosphatase activities during induced granulocytic and monocytic differentiation of HL-60 leukemia cells

About 1.5% of phosphorylated amino acid residues of HL-60 promyelocytic leukemia cells are phosphotyrosine. Induction of granulocytic differentiation by exposure to dimethylsulfoxide decreased tyrosine phosphorylation to 0.2%. A maximum 3-fold increase in tyrosine kinase activity and a 7-fold increase in protein phosphotyrosine phosphatase activity accompanied this change. Monocytic differentiation induced by 12-O-tetradecanoylphorbol-13-acetate, caused a decrease in phosphotyrosine levels to 0.1%; tyrosine kinase activity maximally increased 2-fold, and protein phosphotyrosine phosphatase activity increased 11-fold in these differentiated cells. Thus, although total tyrosine kinase activity markedly increased during differentiation, this was counteracted by an even greater elevation in protein phosphotyrosine phosphatase activity. The findings support the concept that tyrosine phosphorylation is important in the regulation of growth and differentiation of leukemia cells.     

The bcr-c-abl tyrosine kinase activity is extinguished by TPA in K562 leukemia cells

Tyrosine kinase activity is associated with the transforming potential of several oncogenes. Human chronic myeloid leukemia (CML) cells and cell lines have been shown to contain an active bcr-c-abl p210 tyrosine kinase as a consequence of the Philadelphia chromosomal translocation. In the present work the activity of the c-abl and c-src oncogene-encoded tyrosine kinase was investigated during phorbol diester (TPA) induced differentiation of the K562 CML cells. The high tyrosine kinase activity of p210bcr-c-abl is strongly reduced during the initial 24 h of TPA treatment. In contrast, the activity of the c-src tyrosine kinase is not changed. No change occurs in the expression of the c-abl-specific RNAs during this period. Following the reduction of bcr-c-abl kinase activity, cell proliferation is arrested and megakaryoblastic antigens appear on the cells. Sodium butyrate caused a slight decrease in growth rate and of bcr-c-abl kinase activity during erythroid differentiation whereas no changes in c-src or c-abl tyrosine kinase activities were seen in DMSO-treated control cells.     

Protein tyrosine phosphatase profiling analysis of HIB-1B cells during brown adipogenesis

A number of evidence have been accumulated that the regulation of reversible tyrosine phosphorylation, which can be regulated by the combinatorial activity of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), plays crucial roles in various biological processes including differentiation. There are a total of 107 PTP genes in the human genome, collectively referred to as the "PTPome." In this study, we performed PTP profiling analysis of the HIB-1B cell line, a brown preadipocyte cell line, during brown adipogenesis. Through RT-PCR and real-time PCR, several PTPs showing differential expression pattern during brown adipogenesis were identified. In the case of PTP-RE, it was shown to decrease significantly until 4 days after brown adipogenic differentiation, followed by a dramatic increase at 6 days. The overexpression of PTP-RE led to decreased brown adipogenic differentiation via reducing the tyrosine phosphorylation of the insulin receptor, indicating that PTP-RE functions as a negative regulator at the early stage of brown adipogenesis.     

RPTPμ tyrosine phosphatase promotes adipogenic differentiation via modulation of p120 catenin phosphorylation

Adipocyte differentiation can be regulated by the combined activity of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). In particular, PTPs act as key regulators in differentiation-associated signaling pathways. We recently found that receptor-type PTPμ (RPTPμ) expression is markedly increased during the adipogenic differentiation of 3T3-L1 preadipocytes and mesenchymal stem cells. Here, we investigate the functional roles of RPTPμ and the mechanism of its involvement in the regulation of signal transduction during adipogenesis of 3T3-L1 cells. Depletion of endogenous RPTPμ by RNA interference significantly inhibited adipogenic differentiation, whereas RPTPμ overexpression led to an increase in adipogenic differentiation. Ectopic expression of p120 catenin suppressed adipocyte differentiation, and the decrease in adipogenesis by p120 catenin was recovered by introducing RPTPμ. Moreover, RPTPμ induced a decrease in the cytoplasmic p120 catenin expression by reducing its tyrosine phosphorylation level, consequently leading to enhanced translocation of Glut-4 to the plasma membrane. On the basis of these results, we propose that RPTPμ acts as a positive regulator of adipogenesis by modulating the cytoplasmic p120 catenin level. Our data conclusively demonstrate that differentiation into adipocytes is controlled by RPTPμ, supporting the utility of RPTPμ and p120 catenin as novel target proteins for the treatment of obesity.     

On the mechanism of a radiation-induced change in enzymic differentiation during development

In experiments on glucose-6-phosphatase and tyrosine aminotransferase it was shown that radiation induces changes in enzymic differentiation in perinatal rat liver. A study was made of the probable reasons for the observed changes. It was shown that the macromolecular system of the protein enzyme synthesis was not damaged by the radiation doses used. The observed decrease in glucose-6-phosphatase activity during late embryogenesis, after pre-irradiation at early organogenesis, is eliminated by administration of exogenous thyroxine. A radiation-induced rise in the tyrosine aminotransferase activity during the perinatal period correlated with the cyclic AMP system status. It is proposed that modification of enzymic differentiation after irradiation results from the change in the amount of inductors.     

Pim-1 negatively regulates the activity of PTP-U2S phosphatase and influences terminal differentiation and apoptosis of monoblastoid leukemia cells

The levels of Pim-1, a serine/threonine kinase, increase during phorbol myristate acetate (PMA)-induced myeloid cell differentiation. The tyrosine phosphatase PTP-U2S is also associated with PMA-induced differentiation of myeloid cells and has been shown to enhance differentiation and the onset of apoptosis. PTP-U2S contains a Pim-1 phosphorylation consensus sequence, KKRKLTN, which is efficiently phosphorylated by Pim-1. Immunoprecipitated PTP-U2S from U937 cells was phosphorylated by recombinant Pim-1, resulting in a decrease in its phosphatase activity. During PMA-induced differentiation, U937 cells transfected with the dominant negative Pim-1 underwent rapid differentiation and accelerated apoptosis. The opposite effect was observed for wild-type Pim-1. Our results, therefore, provide compelling evidence that Pim-1 functions to negatively regulate PMA-induced differentiation in part through the phosphorylation of PTP-U2S. Together these data suggest that Pim-1 phosphorylates PTP-U2S in vivo to decrease the phosphatase activity that may be necessary to prevent the premature onset of apoptosis following differentiation.     

Initiation of human myoblast differentiation via dephosphorylation of Kir2.1 K+ channels at tyrosine 242

Myoblast differentiation is essential to skeletal muscle formation and repair. The earliest detectable event leading to human myoblast differentiation is an upregulation of Kir2.1 channel activity, which causes a negative shift (hyperpolarization) of the resting potential of myoblasts. After exploring various mechanisms, we found that this upregulation of Kir2.1 was due to dephosphorylation of the channel itself. Application of genistein, a tyrosine kinase inhibitor, increased Kir2.1 activity and triggered the differentiation process, whereas application of bpV(Phen), a tyrosine phosphatase inhibitor, had the opposite effects. We could show that increased Kir2.1 activity requires dephosphorylation of tyrosine 242; replacing this tyrosine in Kir2.1 by a phenylalanine abolished inhibition by bpV(Phen). Finally, we found that the level of tyrosine phosphorylation in endogenous Kir2.1 channels is considerably reduced during differentiation when compared with proliferation. We propose that Kir2.1 channels are already present at the membrane of proliferating, undifferentiated human myoblasts but in a silent state, and that Kir2.1 tyrosine 242 dephosphorylation triggers differentiation.     

PC12 cell neuronal differentiation is associated with prolonged p21ras activity and consequent prolonged ERK activity.

Expression of oncogenic ras in PC12 cells causes neuronal differentiation and sustained protein tyrosine phosphorylation and activity of extracellular signal-regulated kinases (ERKs), p42erk2 and p44erk1. Oncogenic N-ras-induced neuronal differentiation is inhibited by compounds that block ERK protein tyrosine phosphorylation or ERK activity, indicating that ERKs are not only activated by p21ras but serve as the primary downstream effectors of p21ras. Treatment of PC12 cells with nerve growth factor or fibroblast growth factor results in neuronal differentiation and in a sustained elevation of p21ras activity, of ERK activity, and of ERK tyrosine phosphorylation. Epidermal growth factor, which does not cause neuronal differentiation, stimulates only transient (< 1 hr) activation of p21ras and ERKs. These data indicate that transient activation of p21ras and, consequently, ERKs is not sufficient for induction of neuronal differentiation. Prolonged ERK activity is required: a consequence of sustained activation of p21ras by the growth factor receptor protein tyrosine kinase.     

Tyrosine kinase inhibitors reverse butyrate stimulation of human Caco-2 intestinal epithelial cell alkaline phosphatase but not butyrate promotion of dipeptidyl dipeptidase

Short chain fatty acids such as sodium butyrate are concentrated in the colonic lumen and may protect against colon carcinogenesis by maintaining colonocytic differentiation, but the mechanisms by which they act are not fully understood. It has recently been suggested that short chain fatty acids modulate cellular tyrosine kinase activity in addition to altering chromatin structure via regulation of histone acetylation and DNA methylation. Therefore, the authors evaluated the influence of tyrosine kinase inhibition on the effects of 10 mM butyrate on human Caco-2 intestinal epithelial differentiation, using alkaline phosphatase and dipeptidyl dipeptidase specific activity as markers of differentiation, and two tyrosine kinase inhibitors, of different mechanisms of action and different effects on Caco-2 brush border enzyme specific activity, to block tyrosine kinase activity. As expected, butyrate stimulated both alkaline phosphatase and dipeptidyl dipeptidase specific activity. The tyrosine kinase inhibitors prevented, and indeed one inhibitor reversed the effects of butyrate on alkaline phosphatase specific activity. However, tyrosine kinase inhibition did not prevent butyrate stimulation of dipeptidyl dipeptidase specific activity. Different pathways are likely to regulate the effects of butyrate on expression of these two brush border enzymes. Butyrate stimulation of alkaline phosphatase, but not dipeptidyl dipeptidase, may involve tyrosine phosphorylation signaling.     

Protein tyrosine kinase activity in human malaria parasite Plasmodium falciparum.

Protein tyrosine kinases (PTKs) are believed to be implicated in the parasite growth, maturation and differentiation functions. Protein tyrosine kinase activity was found to be distributed in all the stages of P. falciparum parasite maturation. Membrane bound PTK activity was found to be increased during maturation process (ring stage to trophozoite stage) in chloroquine sensitive strains. In vivo conversion of the schizont stage to ring stage via release of merozoites was associated with a decrease in PTK activity. Chloroquine inhibited the membrane bound PTK activity in a dose dependent manner (IC50 = 45 microM). Kinetic studies show that chloroquine is a competitive inhibitor of PTK with respect to peptide substrate and noncompetitive with respect to ATP indicating that chloroquine inhibits PTK activity by binding with protein substrate binding site. The results suggest that maturation of malaria parasite is related to PTK and inhibition of this activity by chloroquine could provide a hypothesis to explain the mechanism of action of chloroquine.     

1alpha, 25-dihydroxy-vitamin D3 alters Syk activation through FcgammaRII in monocytic THP-1 cells

In monocytes and macrophages, activation of the tyrosine kinase Syk is an essential step in the biochemical cascade linking aggregation of receptors for immunoglobulin G (FcgammaR) to initiation of effector functions. An increase in Syk activation during differentiation of myeloid cells by different agents has been reported. We studied the activation state of Syk in response to FcgammaRII crosslinking in monocytic cells before and after in vitro differentiation with 1alpha, 25-dihydroxy-vitamin D3. We show here that while in undifferentiated THP-1 cells clustering of FcgammaRII induces significant phosphorylation and activation of Syk, in THP-1 cells differentiated in vitro by 1alpha, 25-dihydroxy-vitamin D3, FcgammaRII crosslinking induced a decrease in Syk activity. In vitro differentiation did not induce changes in the expression of FcgammaRII isoforms. The observed effect on Syk activation though FcgammaRII could be mediated by differentiation-induced changes in the expression and basal activation level of Syk, as well as changes in the association of Syk with the tyrosine phosphatase SHP-1. These results suggest that the biochemical signaling pathways induced by FcgammaRII could be dependent on the differentiation state of the cell.     

Protein tyrosine phosphatase TbPTP1: A molecular switch controlling life cycle differentiation in trypanosomes

Differentiation in African trypanosomes (Trypanosoma brucei) entails passage between a mammalian host, where parasites exist as a proliferative slender form or a G0-arrested stumpy form, and the tsetse fly. Stumpy forms arise at the peak of each parasitaemia and are committed to differentiation to procyclic forms that inhabit the tsetse midgut. We have identified a protein tyrosine phosphatase (TbPTP1) that inhibits trypanosome differentiation. Consistent with a tyrosine phosphatase, recombinant TbPTP1 exhibits the anticipated substrate and inhibitor profile, and its activity is impaired by reversible oxidation. TbPTP1 inactivation in monomorphic bloodstream trypanosomes by RNA interference or pharmacological inhibition triggers spontaneous differentiation to procyclic forms in a subset of committed cells. Consistent with this observation, homogeneous populations of stumpy forms synchronously differentiate to procyclic forms when tyrosine phosphatase activity is inhibited. Our data invoke a new model for trypanosome development in which differentiation to procyclic forms is prevented in the bloodstream by tyrosine dephosphorylation. It may be possible to use PTP1B inhibitors to block trypanosomatid transmission.     

RhoA/Rho kinase blocks muscle differentiation via serine phosphorylation of insulin receptor substrate-1 and -2

Although the RhoA/Rho kinase (RhoA/ROK) pathway has been extensively investigated, its roles and downstream signaling pathways are still not well understood in myogenic processes. Therefore, we examined the effects of RhoA/ROK on myogenic processes and their signaling molecules using H9c2 and C2C12 cells. Increases in RhoA/ROK activities and serine phosphorylation levels of insulin receptor substrate (IRS)-1 (Ser307 and Ser636/639) and IRS-2 were found in proliferating myoblasts, whereas IRS-1/2 tyrosine phosphorylation and phosphatidylinositol (PI) 3-kinase activity increased during the differentiation process. ROK strongly bound to IRS-1/2 in proliferation medium but dissociated from them in differentiation medium (DM). ROK inactivation by a ROK inhibitor, Y27632, or a dominant-negative ROK, decreased IRS-1/2 serine phosphorylation with increases in IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity, which led to muscle differentiation even in proliferation medium. Inhibition of ROK also enhanced differentiation in DM. ROK activation by a constitutive active ROK blocked muscle differentiation with the increased IRS-1/2 serine phosphorylation, followed by decreases in IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity in DM. Interestingly, fibroblast growth factor-2 added to DM also blocked muscle differentiation through RhoA/ROK activation. Fibroblast growth factor-2 blockage of muscle differentiation was reversed by Y27632. Collectively, these results suggest that the RhoA/ROK pathway blocks muscle differentiation by phosphorylating IRS proteins at serine residues, resulting in the decreased IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity. The absence of the inhibitory effects of RhoA/ROK in DM due to low concentrations of myogenic inhibitory growth factors seems to allow IRS-1/2 tyrosine phosphorylation, which stimulates muscle differentiation via transducing normal myogenic signaling.     

Low molecular weight protein-tyrosine phosphatase is involved in growth inhibition during cell differentiation

Low molecular weight protein-tyrosine phosphatase (LMW-PTP) is an enzyme involved in mitogenic signaling and cytoskeletal rearrangement after platelet-derived growth factor (PDGF) stimulation. Recently, we demonstrated that LMW-PTP is regulated by a redox mechanism involving the two cysteine residues of the catalytic site, which turn reversibly from reduced to oxidized state after PDGF stimulation. Since recent findings showed a decrease of intracellular reactive oxygen species in contact inhibited cells and a lower tyrosine phosphorylation level in dense cultures in comparison to sparse ones, we studied if the level of endogenous LMW-PTP is regulated by growth inhibition conditions, such as cell confluence and differentiation. Results show that both cell confluence and cell differentiation up-regulate LMW-PTP expression in C2C12 and PC12 cells. We demonstrate that during myogenesis LMW-PTP is regulated at translational level and that the protein accumulates at the plasma membrane. Furthermore, we showed that both myogenesis and cell-cell contact lead to a dramatic decrease of tyrosine phosphorylation level of PDGF receptor. In addition, we observed an increased association of the receptor with LMW-PTP during myogenesis. Herein, we demonstrate that myogenesis decreases the intracellular level of reactive oxygen species, as observed in dense cultures. As a consequence, LMW-PTP turns from oxidized to reduced form during muscle differentiation, increasing its activity in growth inhibition conditions such as differentiation. These data suggest that LMW-PTP plays a crucial role in physiological processes, which require cell growth arrest such as confluence and differentiation.     

Effects of modulation of tyrosine phosphorylation on brush border enzyme activity in human Caco-2 intestinal epithelial cells

Intestinal epithelial cell differentiation is closely regulated during normal cell renewal, maturation, and malignant transformation. Since tyrosine phosphorylation influences differentiation in other cell types and has been reported to vary between crypt cells to differentiated villus tip cells, we investigated the influence of tyrosine phosphorylation in colonocyte differentiation, by using human colonic Caco-2 cells as a model and expression of the brush border enzymes alkaline phosphatase (AKP) and dipeptidyl peptidase (DPDD) as differentiation markers. We studied three tyrosine kinase inhibitors with different modes of action and specificities, viz., genistein, erbstatin analog (EA), and tyrphostin, and the tyrosine phosphatase inhibitor sodium orthovanadate. AKP- and DPDD-specific activities were assayed in protein-matched cell lysates by synthetic substrate digestion. We also correlated the effects of these agents on brush border enzyme activity with tyrosine phosphorylation of phosphoproteins by Western blotting. Genistein (5–75?mg/ml) dose-dependently stimulated AKP and DPDD with a maximal stimulation at 75?mg/ml by 158.6± 17.5% and 228.6±37.1% of control values, respectively (n=12, P<0.001). The inactive analog genistin had no effect. Tyrphostin (25?mM) similarly stimulated AKP and DPDD by 138.6±6.6% and 131.8±1.5% of control values (n=12, P<0.001). Unexpectedly, EA (0.1–10?mM) had the opposite effect, inhibiting AKP- and DPDD-specific activity significantly at 10?mM with a maximal 14.8±6.4% and 26.5±2.5% of control values (n=12, each P<0.001). Sodium orthovanadate had a discordant effect on these two differentiation markers. Orthovanadate dose-dependently increased AKP to a maximal 188.5±16.1% of basal activity at 1.5?mM but decreased DPDD activity at 1.5?mM to 47.2±3.8% (n=9, P<0.001 each). The effects of each agent were preserved when proliferation was blocked with mitomycin C, suggesting that the modulation of phenotype by these agents was independent of any effects of proliferation. The tyrosine phosphorylation of several phosphoprotein bands was affected differently by these agents. In particular, the tyrosine phosphorylation of one 70-kDa to 71-kDa band was increased by genistein and tyrophostin but deceased by EA. The different effects of these modulators of tyrosine kinase activity raise the possibility that at least two independent enzymes or pathways regulating tyrosine phosphorylation modulate intestinal epithelial differentiation. Furthermore, tyrosine phosphorylation of the 70-kDa to 71-kDa phosphoprotein may be important in the intracellular signaling by which intestinal epithelial cell differentiation is controlled.     

K562 leukemia cells transfected with the human c-fes gene acquire the ability to undergo myeloid differentiation

Expression of the proto-oncogene p93c-fes and its associated tyrosine kinase activity is marked in mature granulocytes, monocytes, differentiated HL-60 leukemia cells, and leukemia cell lines KG-1, THP-1, HEL, and U-937, which can be induced to differentiate along the granulocyte/monocyte pathway. Conversely, p93-c-fes expression is absent in the K562 cell line, which is resistant to myeloid differentiation. Upon transfection and clonal selection of K562 cells using a mammalian expression vector containing the 13-kilobase pair c-fes gene, c-fes mRNA was transcribed and p93-c-fes tyrosine activity kinase was expressed. Clones expressing c-fes underwent myeloid differentiation as assessed by the appearance of phagocytic activity, Fc receptors, nitro blue tetrazolium reduction, Mac-1 immunofluorescence, and lysozyme production. These results indicate that the expression of the c-fes protooncogene and its associated tyrosine kinase activity plays a major role in the initiation of myeloid differentiation.     

Focal adhesion kinase tyrosine phosphorylation is associated with myogenesis and modulated by insulin

Focal adhesion kinase (FAK) was heavily phosphorylated as a function of differentiation of C2C12 mouse skeletal muscle cells. Insulin caused increases in FAK phosphorylation before stabilization in proliferated cells, while in differentiated cells there was a consistent transient inhibition of FAK phosphorylation before stimulation. The expression level of FAK was unaltered. Specific inhibition of insulin receptor tyrosine kinase activity abolished the insulin-mediated dephosphorylation of FAK. The data strongly indicate that FAK tyrosine phosphorylation, necessary for skeletal muscle differentiation, is modulated by insulin. Thus, for the first time, we report the differential regulation of FAK tyrosine phosphorylation by insulin during skeletal muscle differentiation.     

The human p50csk tyrosine kinase phosphorylates p56lck at Tyr-505 and down regulates its catalytic activity.

Protein tyrosine kinases participate in the transduction and modulation of signals that regulate proliferation and differentiation of cells. Excessive or deregulated protein tyrosine kinase activity can cause malignant transformation. The catalytic activity of the T cell protein tyrosine kinase p56lck is normally suppressed by phosphorylation of a carboxyl-terminal tyrosine, Tyr-505, by another cellular protein tyrosine kinase. Here we characterize a human cytosolic 50 kDa protein tyrosine kinase, p50csk, which specifically phosphorylates Tyr-505 of p56lck and a synthetic peptide containing this site. Phosphorylation of Tyr-505 suppressed the catalytic activity of p56lck. We suggest that p50csk negatively regulates p56lck, and perhaps other cellular src family kinases.     

Butyrate-induced G2/M block in Caco-2 colon cancer cells is associated with decreased p34cdc2 activity

Butyrate, a short-chain fatty acid, has been reported to inhibit proliferation and stimulate differentiation in multiple cancer cell lines. Whereas the effects of butyrate on cellular differentiation are well documented, the relationship between butyrate-induced differentiation and its effect on cell cycle traverse is less well understood. The purpose of this study was to investigate the effects of butyrate on the regulatory proteins of the G2/M traverse in the Caco-2 colon cancer cell model. We demonstrated that the inhibition of proliferation and increased cellular differentiation after treatment of Caco-2 cells with butyrate were associated with a significant G2/M cell cycle block. Although protein levels of the major G2/M regulatory protein, p34cdc2, were unchanged, a decrease in p34cdc2 activity was noted. Despite this decrease in activity, the inhibitory tyrosine phosphorylation of p34cdc2 was decreased, suggesting that other factors are responsible for the decreased kinase activity. The reduced activity of p34cdc2 provides a possible mechanism for the accumulation of Caco-2 cells in the G2/M cell cycle compartment following exposure to butyrate. This cell system provides a new model for studies of G2/M cell cycle perturbations.     

Synergistic induction of erythroid differentiation of mouse erythroleukemia (MEL) cells by inhibitors of topoisomerases and protein tyrosine kinases.

In vitro erythroid differentiation of mouse erythroleukemia (MEL) cells was induced by combinations of topoisomerase and protein kinase inhibitors. Neither inhibitor alone exhibited inducing activity. Although inhibitors of topoisomerases I and II were equally effective in the synergistic induction of erythroid differentiation, only inhibitors of tyrosine kinases, not of serine/threonine kinases, exhibited synergistic activity. The erythroid differentiation induced by the combination of topoisomerase and protein tyrosine kinase inhibitors was distinguished from that induced by typical erythroid inducing agents such as DMSO or HMBA by (1) earlier hemoglobin accumulation in the cells and (2) insensitivity to specific inhibitors (dexamethasone and sodium orthovanadate) of MEL cell differentiation.