Tyrosine phosphorylation in human lymphomas

In a previous study, we showed that the high level of protein tyrosine phosphorylation present in lymphomas containing an anaplastic lymphoma kinase (ALK) can be demonstrated in routinely processed paraffin tissue sections using immunolabelling techniques. In the present study we investigated whether oncogenic tyrosine kinase activation also occurs in other categories of lymphoma by staining 145 cases of lymphoma covering those tumours with a range of different subtypes including those with morphological similarity to ALK-positive anaplastic large cell lymphoma (ALCL). Twelve cases of the borderline malignant disorder lymphomatoid papulosis were also studied. Twenty seven of the 28 cases of ALK-positive ALCL showed the extensive cytoplasmic labelling for phosphotyrosine in the neoplastic cells. The remaining case containing moesin-ALK exhibited membrane-associated phosphotyrosine expression. There was no nuclear phosphotyrosine labelling in any of the ALK-positive ALCL, even though ALK was present within the cell nuclei in 23 of the tumours. Variable degrees of phosphotyrosine labelling, usually membrane-restricted, were observed in 7/40 cases of ALK-negative ALCL, 9/29 cases of diffuse large B-cell lymphoma, 3/6 cases of mediastinal B-cell lymphoma, 2/7 cases of Hodgkin's lymphoma, 3/6 cases of peripheral T-cell lymphomas unspecified, 4/6 cases of B-cell chronic lymphocytic leukaemia, 2/6 cases of follicular lymphomas and 2/12 cases of lymphomatoid papulosis studied. However none of these phosphotyrosine-positive cases showed the strong cytoplasmic labelling comparable to that seen in ALK-positive lymphoma. We conclude that activation of a tyrosine kinase is probably not a major oncogenic event in lymphomas other than ALK-positive ALCL.     

Tyrosine phosphorylation in human lymphomas

In a previous study, we showed that the high level of protein tyrosine phosphorylation present in lymphomas containing an anaplastic lymphoma kinase (ALK) can be demonstrated in routinely processed paraffin tissue sections using immunolabelling techniques. In the present study we investigated whether oncogenic tyrosine kinase activation also occurs in other categories of lymphoma by staining 145 cases of lymphoma covering those tumours with a range of different subtypes including those with morphological similarity to ALK-positive anaplastic large cell lymphoma (ALCL). Twelve cases of the borderline malignant disorder lymphomatoid papulosis were also studied. Twenty seven of the 28 cases of ALK-positive ALCL showed the extensive cytoplasmic labelling for phosphotyrosine in the neoplastic cells. The remaining case containing moesin-ALK exhibited membrane-associated phosphotyrosine expression. There was no nuclear phosphotyrosine labelling in any of the ALK-positive ALCL, even though ALK was present within the cell nuclei in 23 of the tumours. Variable degrees of phosphotyrosine labelling, usually membrane-restricted, were observed in 7/40 cases of ALK-negative ALCL, 9/29 cases of diffuse large B-cell lymphoma, 3/6 cases of mediastinal B-cell lymphoma, 2/7 cases of Hodgkin's lymphoma, 3/6 cases of peripheral T-cell lymphomas unspecified, 4/6 cases of B-cell chronic lymphocytic leukaemia, 2/6 cases of follicular lymphomas and 2/12 cases of lymphomatoid papulosis studied. However none of these phosphotyrosine-positive cases showed the strong cytoplasmic labelling comparable to that seen in ALK-positive lymphoma. We conclude that activation of a tyrosine kinase is probably not a major oncogenic event in lymphomas other than ALK-positive ALCL.     

Tyrosine phosphorylation of the human guanylyl cyclase C receptor

Tyrosine phosphorylation events are key components of several cellular signal transduction pathways. This study describes a novel method for identification of substrates for tyrosine kinases. Co-expression of the tyrosine kinase EphB1 with the intracellular domain of guanylyl cyclase C (GCC) inEscherichia coli cells resulted in tyrosine phosphorylation of GCC, indicating that GCC is a potential substrate for tyrosine kinases. Indeed, GCC expressed in mammalian cells is tyrosine phosphorylated, suggesting that tyrosine phosphorylation may play a role in regulation of GCC signalling. This is the first demonstration of tyrosine phosphorylation of any member of the family of membrane-associated guanylyl cyclases.     

Tyrosine phosphorylation in brassinosteroid signaling

Brassinosteroids (BRs) regulate plant growth and development through a complex signal transduction pathway involving BRASSINOSTEROID INSENSITIVE 1 (BRI1), which is the BR receptor, and its co-receptor BRI1-ASSOCIATED KINASE 1 (BAK1). Both proteins are classified as Ser/Thr protein kinases. Recently, we reported that recombinant cytoplasmic domains (CD) of BRI1 and BAK1 also autophosphorylate on tyrosine residues and thus are dual-specificity kinases.¹ Two sites of Tyr autophosphorylation were identified that appear to have different effects on BRI1 function. Tyr-831 in the juxtamembrane domain is not essential for kinase activity but has a regulatory role, with phosphorylation of Tyr-831 causing inhibition of growth and delay of flowering. In contrast, Tyr-956 is located in subdomain IV of the kinase domain and is essential for kinase activity, and we are speculating that the free hydroxyl group at this position is essential and thus phosphorylation of Tyr-956 would inhibit BRI1 kinase activity. Expression of BRI1(Y831F)-Flag in the weak allele bri1-5 rescued the dwarf phenotype but plants had rounder leaves, increased shoot biomass, and flowered earlier than plants expressing the BRI1(wild type)-Flag in the bri1-5 background. To further elaborate on earlier results, we present additional phenotypic analysis of transgenic Arabidopsis plants expressing BRI1(Y831F)-Flag or site-directed mutants of other Tyr residues within the kinase domain. The results highlight the unique role of Tyr-831 in regulation of BR signaling in vivo. Elucidating the molecular basis for increased biomass accumulation in plants expressing BRI1(Y831F)-Flag may have applications for agriculture.Key words: brassinosteroids, LRR-RLK, autophosphorylation, tyrosine phosphorylation, signal transduction     

Tyrosine phosphorylation profiling in FGF-2 stimulated human embryonic stem cells

The role of fibroblast growth factor-2 (FGF-2) in maintaining undifferentiated human embryonic stem cells (hESC) was investigated using a targeted phosphoproteomics approach to specifically profile tyrosine phosphorylation events following FGF-2 stimulation. A cumulative total number of 735 unique tyrosine phosphorylation sites on 430 proteins were identified, by far the largest inventory to date for hESC. Early signaling events in FGF-2 stimulated hESC were quantitatively monitored using stable isotope dimethyl labeling, resulting in temporal tyrosine phosphorylation profiles of 316 unique phosphotyrosine peptides originating from 188 proteins. Apart from the rapid activation of all four FGF receptors, trans-activation of several other receptor tyrosine kinases (RTKs) was observed as well as induced tyrosine phosphorylation of downstream proteins such as PI3-K, MAPK and several Src family members. Both PI3-K and MAPK have been linked to hESC maintenance through FGF-2 mediated signaling. The observed activation of the Src kinase family members by FGF-2 and loss of pluripotent marker expression post Src kinase inhibition may point to the regulation of cytoskeletal and actin depending processes to maintain undifferentiated hESC.     

Tyrosine phosphorylation of interleukin 2 receptor-related proteins in phytohemagglutinin-activated human lymphocytes

Three classes of proteins (mol wts 70k, 64k and 45k) having the characteristics of interleukin 2 receptor were detected in phytohemagglutinin-activated human lymphocytes using two monoclonal antibodies which recognize distinct epitopes on the receptor. It was shown that at least portions of these proteins were phosphorylated on tyrosine by analyses for phosphotyrosine by immunoblotting and by immunoaffinity chromatography with antibodies to phosphotyrosine. In addition an iodinated phosphotyrosine derivative was identified in partial hydrolysates of these proteins iodinated in vitro.     

Tyrosine phosphorylation in Toll-like receptor signaling

There is a wealth of knowledge about how different Ser/Thr protein kinases participate in Toll-like receptor (TLR) signaling. In many cases, we know the identities of the Ser/Thr residues of various components of the TLR-signaling pathways that are phosphorylated, the functional consequences of the phosphorylation and the responsible protein kinases. In contrast, the analysis of Tyr-phosphorylation of TLRs and their signaling proteins is currently incomplete, because several existing analyses are not systematic or they do not rely on robust experimental data. Nevertheless, it is clear that many TLRs require, for signaling, ligand-dependent phosphorylation of specific Tyr residues in their cytoplasmic domains; the list includes TLR2, TLR3, TLR4, TLR5, TLR8 and TLR9. In this article, we discuss the current status of knowledge of the effect of Tyr-phosphorylation of TLRs and their signaling proteins on their biochemical and biological functions, the possible identities of the relevant protein tyrosine kinases (PTKs) and the nature of regulations of PTK-mediated activation of TLR signaling pathways.     

Tyrosine phosphorylation of WW proteins

A number of key regulatory proteins contain one or two copies of the WW domain known to mediate protein–protein interaction via proline-rich motifs, such as PPxY. The Hippo pathway components take advantage of this module to transduce tumor suppressor signaling. It is becoming evident that tyrosine phosphorylation is a critical regulator of the WW proteins. Here, we review the current knowledge on the involved tyrosine kinases and their roles in regulating the WW proteins.     

Tyrosine phosphorylation of plant tubulin

Phosphorylation of αβ-tubulins dimers by protein tyrosine kinases plays an important role in the regulation of cellular growth and differentiation in animal cells. In plants, however, the role of tubulin tyrosine phosphorylation is unknown and data on this tubulin modification are limited. In this study, we used an immunochemical approach to demonstrate that tubulin isolated by both immunoprecipitation and DEAE-chromatography is phosphorylated on tyrosine residues in cultured cells of Nicotiana tabacum. This opens up the possibility that tyrosine phosphorylation of tubulin could be involved in modulating the properties of plant microtubules.     

Tyrosine phosphorylation of alpha-actinin in activated platelets

The integrin alpha(IIb)beta(3) mediates tyrosine phosphorylation of a 105-kDa protein (pp105) in activated platelets. We have partially purified a 105-kDa tyrosine-phosphorylated protein from platelets stimulated with phorbol 12-myristate 13-acetate and obtained the sequence of an internal 12-mer peptide derived from this protein. The sequence was identical to human alpha-actinin sequences deposited in the Swiss Protein Database. alpha-Actinin, a 105-kDa protein in platelets, was subsequently purified from activated platelets by four sequential chromatographic steps. Fractions were analyzed by Western blotting and probed with alpha-actinin and anti-phosphotyrosine antibodies. The distribution of alpha-actinin and pp105 overlapped throughout the purification. Furthermore, in the course of this purification, a 105-kDa tyrosine-phosphorylated protein was only detected in fractions that contained alpha-actinin. The purified alpha-actinin protein was immunoprecipitated with antibodies to phosphotyrosine in the absence but not in the presence of phenyl phosphate. alpha-Actinin resolved by two-dimensional gel electrophoresis of activated platelet lysates was recognized by the antibodies to phosphotyrosine, whereas pretreatment of the platelets with bisindolylmaleimide, a protein kinase C inhibitor that prevents tyrosine phosphorylation of pp105, inhibited the reactivity of the antibodies to phosphotyrosine with alpha-actinin. Taken together, these data demonstrate that a fraction of alpha-actinin is tyrosine-phosphorylated in activated platelets.     

Tyrosine phosphorylation is involved in Ca(2+)entry in human gingival fibroblasts

Bradykinin (1 microM) and histamine (100 microM) evoked an initial transient increase and a subsequent sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded human gingival fibroblasts, which may be attributed to Ca(2+) release from intracellular stores and Ca(2+) entry from extracellular sites, respectively. In fibroblasts pretreated with tyrosine kinase inhibitors such as herbimycin A (1 microM) and tyrphostin 47 (20 microM), the sustained level of [Ca(2+)](i) induced by bradykinin and histamine increased, but not the initial peak level. In the absence of external Ca(2+), bradykinin and histamine induced only the transient increase in [Ca(2+)](i), but a subsequent addition of Ca(2+) to the medium resulted in a sustained increase in [Ca(2+)](i) caused by Ca(2+)entry. Thapsigargin, an inhibitor of Ca(2+)-ATPase in inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores, mimicked the effect of bradykinin and histamine. In the fibroblasts pretreated with tyrosine kinase inhibitors, the bradykinin-, histamine- and thapsigargin-induced Ca(2+) entry was clearly enhanced, but not the transient [Ca(2+)](i) increase. Tyrosine phosphatase inhibitor benzylphosphonic acid (200 microM) had no effect on Ca(2+)entry or transient [Ca(2+)](i) increase. These results suggest that tyrosine phosphorylation is involved in Ca(2+) entry in human gingival fibroblasts.     

Tyrosine phosphorylation of the asialoglycoprotein receptor

The asialoglycoprotein (ASGP) receptor undergoes constitutive endocytosis through the coated pit/coated vesicle pathway in hepatocytes. Studies on HepG2 cells have shown that the receptor is phosphorylated at serine under control conditions and following protein kinase C stimulation. This study examined whether the ASGP receptor could also serve as a substrate for a tyrosine kinase in HepG2 cells. 32P labeling was performed in membrane preparations, in permeabilized cells at 4 degrees C, and in intact cells at 37 degrees C. The phosphorylated ASGP receptor was isolated by immunoprecipitation, hydrolyzed in 6 N HCl at 110 degrees C, and analyzed by two-dimensional high voltage electrophoresis. The receptor isolated from a membrane preparation incubated in vitro with [gamma-32P]ATP incorporated radiolabel predominantly (greater than 90%) into phosphotyrosine. ASGP receptor phosphorylation at both tyrosine and serine was detected in intact cells incubated with phosphatase inhibitors for 60 min at 37 degrees C. The presence of both phenylarsine oxide (20 microM) and sodium orthovanadate (200 microM) was required for tyrosine phosphorylation. Use of these inhibitors together resulted in a 16.4-fold increase in phosphorylation of the immunoprecipitated ASGP receptor, whereas phosphorylation of total HepG2 membrane proteins was not significantly augmented by this procedure. Selective proteolytic digestion of ASGP receptors in isolated vesicles demonstrated that the phosphorylation site identified in these studies is located at tyrosine 5 in the cytoplasmic tail.     

Tyrosine phosphorylation of phosphatase inhibitor 2

Inhibitor 2 is a heat-stable protein that complexes with the catalytic subunit of type-1 protein phosphatase. The reversible phosphorylation of Thr 72 of the inhibitor in this complex has been shown to regulate phosphatase activity. Here we show that inhibitor 2 can also be phosphorylated on tyrosine residues. Inhibitor 2 was ³²P-labeled by the insulin receptor kinase in vitro, in the presence of polylysine. Phosphorylation of inhibitor 2 was accompanied by decreased electrophoretic mobility. Dephosphorylation of inhibitor 2 by tyrosine phosphatase 1B, restored normal electrophoretic mobility. Phosphotyrosine in inhibitor 2 was detected by immunoblotting with antiphosphotyrosine antibodies and phosphoamino acid analysis. In addition, following tryptic digestion, one predominant phosphopeptide was recovered at the anode. The ability of inhibitor 2 to inhibit type-1 phosphatase activity was diminished with increasing phosphorylation up to a stoichiometry of 1 mole phosphate incorporated/mole of inhibitor 2, where inhibitory activity was completely lost. These data demonstrate that inhibitor 2 can be phosphorylated on tyrosine residues by the insulin receptor kinase, resulting in a molecule with decreased ability to inhibit type-1 phosphatase activity.     

Calcium ion-dependent adenosine triphosphatase activity and plasma-membrane phosphorylation in the human neutrophil.

A plasma-membrane fraction was isolated from a post-nuclear extract of human neutrophils by centrifugation through a linear sucrose density gradient. This fraction exhibited a Ca2+-dependent adenosine triphosphatase (ATPase) activity that could be differentiated from mitochondrial or myosin ATPase and from plasma-membrane Mg2+-dependent ATPase. When assayed in the presence of [gamma-32P]ATP, the Ca2+-dependent ATPase reaction resulted in the formation of an acid-resistant hydroxylamine-sensitive bond between the gamma-[32P] phosphate group and a membrane protein subunit with an apparent mol.wt. of 135000. Half-maximal activating effect of Ca2+ was found at 82nM and 0.18 microM for the ATPase and the formation of the 32P-membrane complex respectively. Generation of the phosphorylated product attained the steady state at 0 degrees C by about 30s, and was rapidly reversed by ADP. These results suggest that the Ca2+-activated ATPase reaction occurs through the formation of a phosphoprotein intermediate, similar to that described for some Ca2+-dependent ATPase enzymes associated with Ca2+ transport. The possibility thus exists that the neutrophil Ca2+-dependent ATPase catalyses a process of Ca2+ extrusion from the cell, thereby participating in the regulation of several Ca2+-dependent neutrophil functions.     

Tyrosine phosphorylation of caveolin-1 in the endothelium

Caveolin-1, a scaffolding protein of caveolae, is known to be tyrosine-phosphorylated by Src kinases. Recently we generated a specific antibody to caveolin-1 phosphorylated at tyrosine-14 (PY14) (R. Nomura and T. Fujimoto, 1999, Mol. Biol. Cell 10, 975-986). In the present study, by applying PY14 to sections of normal rat tissues, we found that tyrosine phosphorylation of caveolin-1 occurred in limited locations, including the endothelium of the continuous capillaries and small venules. Cultured endothelial cells were not labeled by PY14 under a standard culture condition, but became positively labeled when exposed to oxidative stresses and/or tyrosine phosphatase inhibitors. The reaction was prohibited by pretreating the cells with herbimycin A or genistein. Vasoactive reagents or physical stimuli did not cause the phosphorylation. Concomitant with the tyrosine phosphorylation, the number of invaginated caveolae decreased drastically, and vesicles labeled intensely for caveolin-1 appeared in the cytoplasm; the average diameter of the vesicles was larger than that of caveolae. The result implies that tyrosine phosphorylation of caveolin-1 occurs at tyrosine-14 in the normal rat endothelium in vivo and may induce caveolar vesiculation and/or fusion.     

Tyrosine phosphorylation and invasion mechanisms in Eimeria tenella

The role of signal transduction mechanisms with regard to the host cell invasion mechanics used by apicomplexans appears to have been overlooked: indeed, it is obvious that a signal must be transduced from the surface of an invading parasite to an intracellular location within it once the parasite makes contact with a host cell for the invasion process to be initiated. Data outlined in this communication show strong evidence for the role of tyrosine phosphorylation in the molecular mechanics and control of invasion of host cells when set within the context of the available literature, as detailed in this study.     

Tyrosine phosphorylation of a 66,000 Mr soluble protein in lectin-activated human peripheral blood T lymphocytes

Protein phosphorylation was studied in human T lymphocytes stimulated with the mitogenic lectins phytohemagglutinin (PHA) and concanavalin A (Con A). The T lymphocytes were prepared from the venous blood of normal volunteers, their intracellular ATP pools were labeled with [32P]orthophosphate, and protein phosphorylation was assayed in the soluble fraction by two-dimensional gel electrophoresis and autoradiography. When lymphocytes stimulated with PHA or Con A were compared to unstimulated control cells, there was a general increase in protein phosphorylation and the specific phosphorylation of a soluble protein with Mr = 64.9 to 69 KD and pI = 5.6 to 5.8. Phosphorylation of this protein, designated TPP-66, was observed as early as 2 min after the addition of lectin with a gradual increase in the level of phosphorylation over the next 120 min. In the majority of experiments, there was no phosphorylation seen in the unstimulated lymphocytes; however, in some experiments, there was appreciable phosphorylation, which was seen beginning 60 min after the labeling period. When the TPP-66 spot from stimulated lymphocytes was excised from gels, was eluted, and was subjected to limited base hydrolysis followed by single-dimension high voltage electrophoresis, the major phosphorylated residue migrated with phosphotyrosine. In some experiments, there was phosphorylation of serine residues in both the stimulated and control cells; tyrosine phosphorylation was never seen in the unstimulated cell population. These data suggest that, like other stimuli for cell growth, the induction of lymphocyte growth by lectins is associated with the activation of a tyrosine-specific kinase. Thus, tyrosine phosphorylation may play a key role in the transmission of the signal for lymphocyte growth from the exterior to the interior of the cell.     

Tyrosine hydroxylase phosphorylation: regulation and consequences

The rate-limiting enzyme in catecholamine synthesis is tyrosine hydroxylase. It is phosphorylated at serine (Ser) residues Ser8, Ser19, Ser31 and Ser40 in vitro, in situ and in vivo. A range of protein kinases and protein phosphatases are able to phosphorylate or dephosphorylate these sites in vitro. Some of these enzymes are able to regulate tyrosine hydroxylase phosphorylation in situ and in vivo but the identity of the kinases and phosphatases is incomplete, especially for physiologically relevant stimuli. The stoichiometry of tyrosine hydroxylase phosphorylation in situ and in vivo is low. The phosphorylation of tyrosine hydroxylase at Ser40 increases the enzyme's activity in vitro, in situ and in vivo. Phosphorylation at Ser31 also increases the activity but to a much lesser extent than for Ser40 phosphorylation. The phosphorylation of tyrosine hydroxylase at Ser19 or Ser8 has no direct effect on tyrosine hydroxylase activity. Hierarchical phosphorylation of tyrosine hydroxylase occurs both in vitro and in situ, whereby the phosphorylation at Ser19 increases the rate of Ser40 phosphorylation leading to an increase in enzyme activity. Hierarchical phosphorylation depends on the state of the substrate providing a novel form of control of tyrosine hydroxylase activation.