Inhibitors of tyrosine kinases and phosphatases as a tool for the investigation of microtubule role in plant cold response

Tyrosine phosphorylation plays a vital role in the variety of signal transduction pathways in eukaryotic cells, however its role and relevance in plants are still largely unknown. To investigate the functional role of tubulin tyrosine phosphorylation in plant cells the interplay between the effects of tyrosine kinases (herbimycin A) as well as tyrosine phosphatases (sodium orthova nadate) inhibitors on microtubules sensitivity to cold in A. thaliana root cells were studied. Since it was found that inhibition of tyrosine kinases significantly increased the microtubules sensitivity to cold, while inhibition of tyrosine phophatases enhanced their cold-resistance, we suggest an existence of certain functional interaction between the phosphorylation on tyrosine residues and sensitivity of cortical microtubules to low temperatures.     

Effects of tyrosine kinase and phosphatase inhibitors on microtubules in Arabidopsis root cells

To investigate the role of tyrosine phosphorylation/dephosphorylation processes in plant cells the morphology of Arabidopsis thaliana primary roots and the organization of cortical microtubules (MTs) were studied after inhibition of protein tyrosine kinases (PTKs) and tyrosine phosphatases (PTPs). It was found that all tested types of PTKs inhibitors (herbimycin A, genistein and tyrphostin AG 18) altered root hair growth and development, probably as a result of their significant influences on MTs organization in root hairs. The treatment also led to MTs reorientation and disruption in epidermis and cortex cells of both elongation and differentiation zones of primary roots. Enhanced tyrosine phosphorylation after treatment with a PTPs inhibitor (sodium orthovanadate) resulted in intense induction of root hair development and growth and caused a significant shortening of the elongation zone. It also led to changes of MTs orientation from transverse to longitudinal in epidermis and cortex cells of the elongation and differentiation zones of the root. From the data obtained we can suppose that tyrosine phosphorylation can be involved in the dynamics and organization of MTs in different types of plant cells.     

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.     

Identification of Fer tyrosine kinase localized on microtubules as a platelet endothelial cell adhesion molecule-1 phosphorylating kinase in vascular endothelial cells

Platelet endothelial adhesion molecule-1 (PECAM-1) is a part of intercellular junctions and triggers intracellular signaling cascades upon homophilic binding. The intracellular domain of PECAM-1 is tyrosine phosphorylated upon homophilic engagement. However, it remains unclear which tyrosine kinase phosphorylates PECAM-1. We sought to isolate tyrosine kinases responsible for PECAM-1 phosphorylation and identified Fer as a candidate, based on expression cloning. Fer kinase specifically phosphorylated PECAM-1 at the immunoreceptor tyrosine-based inhibitory motif. Notably, Fer induced tyrosine phosphorylation of SHP-2, which is known to bind to the immunoreceptor tyrosine-based inhibitory motif of PECAM-1, and Fer also induced tyrosine phosphorylation of Gab1 (Grb2-associated binder-1). Engagement-dependent PECAM-1 phosphorylation was inhibited by the overexpression of a kinase-inactive mutant of Fer, suggesting that Fer is responsible for the tyrosine phosphorylation upon PECAM-1 engagement. Furthermore, by using green fluorescent protein-tagged Fer and a time-lapse fluorescent microscope, we found that Fer localized at microtubules in polarized and motile vascular endothelial cells. Fer was dynamically associated with growing microtubules in the direction of cell-cell contacts, where p120catenin, which is known to associate with Fer, colocalized with PECAM-1. These results suggest that Fer localized on microtubules may play an important role in phosphorylation of PECAM-1, possibly through its association with p120catenin at nascent cell-cell contacts.     

Chlamydia trachomatis tarp is phosphorylated by src family tyrosine kinases

The translocated actin recruiting phosphoprotein (Tarp) is injected into the cytosol shortly after Chlamydia trachomatis attachment to a target cell and subsequently phosphorylated by an unidentified tyrosine kinase. A role for Tarp phosphorylation in bacterial entry is unknown. In this study, recombinant C. trachomatis Tarp was employed to identify the host cell kinase(s) required for phosphorylation. Each tyrosine rich repeat of L2 Tarp harbors a sequence similar to a Src and Abl kinase consensus target. Furthermore, purified p60-src, Yes, Fyn, and Abl kinases were able to phosphorylate Tarp. Mutagenesis of potential tyrosines within a single tyrosine rich repeat peptide indicated that both Src and Abl kinases phosphorylate the same residues suggesting that C. trachomatis Tarp may serve as a substrate for multiple host cell kinases. Surprisingly, chemical inhibition of Src and Abl kinases prevented Tarp phosphorylation in culture and had no measurable effect on bacterial entry into host cells.     

Different kinases desensitize the human delta-opioid receptor (hDOP-R) in the neuroblastoma cell line SK-N-BE upon peptidic and alkaloid agonists

In a previous work, we described a differential desensitization of the human δ-opioid receptor (hDOP-R) by etorphine (a non-selective and alkaloid agonist) and δ-selective and peptidic agonists (DPDPE ([d-Pen^2,5]enkephalin) and deltorphin I (Tyr-d-Ala-Phe-Asp-Val-Val-Gly-NH₂)) in the neuroblastoma cell line SK-N-BE (Allouche et al., Eur. J. Pharmacol., 371, 235, 1999). In the present study, we explored the putative role of different kinases in this differential regulation.

First, selective chemical inhibitors of PKA, PKC and tyrosine kinases were used and we showed a significant reduction of etorphine-induced opioid receptor desensitization by the bisindolylmaleimide I (PKC inhibitor) while genistein (tyrosine kinase inhibitor) was potent to impair desensitization induced by the different agonists. When the PKA was inhibited by H89 pretreatment, no modification of opioid receptor desensitization was observed whatever the agonist used.

Second, we further studied the role of G protein-coupled receptor kinases (GRKs) and by using western-blot experiments we observed that only the GRK2 isoform was expressed in the SK-N-BE cells. Next, the neuroblastoma cells were transfected with the wild type GRK2 or its dominant negative mutant GRK2-K220R and the inhibition on cAMP level was determined in naïve and agonist-pretreated cells. We showed that over-expression of GRK2-K220R totally abolished etorphine-induced receptor desensitization while no effect was observed with peptidic agonists and over-expression of GRK2 selectively impaired cAMP inhibition promoted by etorphine suggesting that this kinase was involved in the regulation of hDOP-R activated only by etorphine.

Third, correlation between functional experiments and phosphorylation of the hDOP-R after agonist activation was assessed by western-blot using the specific anti-phospho-DOP-R Ser³⁶³ antibody. While all agonists were potent to increase phosphorylation of opioid receptor, we showed no impairment of receptor phosphorylation level after PKC inhibitor pretreatment. Upon agonist activation, no enhancement of receptor phosphorylation was observed when the GRK2 was over-expressed while the GRK2-K220R partially reduced the hDOP-R Ser³⁶³ phosphorylation only after peptidic agonists pretreatment.

In conclusion, hDOP-R desensitization upon etorphine exposure relies on the GRK2, PKC and tyrosine kinases while DPDPE and deltorphin I mediate desensitization at least via tyrosine kinases. Although the Ser³⁶³ was described as the primary phosphorylation site of the mouse DOP-R, we observed no correlation between desensitization and phosphorylation of this amino acid.     

Src and Pyk2 mediate angiotensin II effects in cultured rat astrocytes

Angiotensin II (Ang II)-induced proliferation of rat astrocytes is mediated by multiple signaling pathways. In the present study, we investigated the role of non-receptor tyrosine kinases on Ang II-signaling and proliferation of astrocytes cultured from neonatal rat pups. Ang II stimulated astrocyte growth, ERK1/2 phosphorylation and the phosphorylation of Src and proline-rich tyrosine kinase-2 (Pyk2), in astrocytes obtained from brainstem and cerebellum. Pretreatment with 10 microM PP2, a selective Src inhibitor, inhibited Ang II stimulated ERK1/2 phosphorylation by 59% to 91% both in brainstem and cerebellum astrocytes. PP2 also inhibited Ang II induction of brainstem (76% inhibition) and cerebellar (64% inhibition) astrocyte growth. Similarly, pretreatment with 25 microM dantrolene, the Pyk2 inhibitor, attenuated ERK1/2 activity in brainstem (62% inhibition) and in cerebellum astrocytes (44% inhibition). Interestingly, inhibition of Pyk2 inhibited Ang II-induced Src activation suggesting that these two non-receptor tyrosine kinases may be acting in concert to mediate Ang II effects in astrocytes. In summary, we found that Ang II stimulates the non-receptor tyrosine kinases Src and Pyk2 which mediate Ang II-induced ERK1/2 activation leading to stimulation of astrocyte growth. In addition, these two tyrosine kinases may be interacting to regulate effects of the peptide in these cells.     

alpha-synuclein is phosphorylated by members of the Src family of protein-tyrosine kinases

alpha-Synuclein (alpha-Syn) is implicated in the pathogenesis of Parkinson's Disease, genetically through missense mutations linked to early onset disease and pathologically through its presence in Lewy bodies. alpha-Syn is phosphorylated on serine residues; however, tyrosine phosphorylation of alpha-Syn has not been established (, ). A comparison of the protein sequence between Synuclein family members revealed that all four tyrosine residues of alpha-Syn are conserved in all orthologs and beta-Syn paralogs described to date, suggesting that these residues may be of functional importance (). For this reason, experiments were performed to determine whether alpha-Syn could be phosphorylated on tyrosine residue(s) in human cells. Indeed, alpha-Syn is phosphorylated within 2 min of pervanadate treatment in alpha-Syn-transfected cells. Tyrosine phosphorylation occurs primarily on tyrosine 125 and was inhibited by PP2, a selective inhibitor of Src protein-tyrosine kinase (PTK) family members at concentrations consistent with inhibition of Src function (). Finally, we demonstrate that alpha-Syn can be phosphorylated directly both in cotransfection experiments using c-Src and Fyn expression vectors and in in vitro kinase assays with purified kinases. These data suggest that alpha-Syn can be a target for phosphorylation by the Src family of PTKs.     

Role of protein kinase activation in the induction of B cell adhesion by MHC class II ligands.

Engagement of MHC class II (Ia) molecules on B cells induces tyrosine phosphorylation, phosphoinositide turnover, elevation of intracellular calcium concentrations, and a rise in cAMP levels. However, a role for these biochemical signals in mediating functional responses induced by Ia ligands remains largely undefined. In this study, we utilized the induction of B cell adhesion by Ia ligands to demonstrate a role for signals transduced via Ia molecules in the generation of a functional response. Ia ligands that induced B cell aggregation induced tyrosine phosphorylation, whereas Ia ligands that did not induce B cell aggregation failed to induce any detectable tyrosine phosphorylation. Ia-induced B cell aggregation and tyrosine phosphorylation were inhibited by genistein and by herbimycin A, inhibitors of tyrosine kinases (PTK). Sphingosine and calphostin C, inhibitors of protein kinase C (PKC), also inhibited Ia-induced adhesion whereas HA1004, an inhibitor of cyclic nucleotide-dependent kinases, did not. Ia ligands induced both LFA-1-dependent and LFA-1-independent B cell adhesion. These two pathways of cell adhesion differed in their requirement for activation signals. PKC activation was sufficient for LFA-1-dependent adhesion, whereas LFA-1-independent adhesion required independent phosphorylation events mediated by PKC and by PTK. These results provide functional relevance for biochemical signals transduced via Ia molecules by demonstrating that Ia-induced B cell adhesion is mediated by the activation of PKC and by one or more PTK.     

Requirement of protein tyrosine kinase and phosphatase activities for human sperm exocytosis

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli, sperm undergo calcium-dependent exocytosis termed the acrosome reaction, which is an absolute prerequisite for fertilization. Protein tyrosine phosphorylation and dephosphorylation are a mechanisms by which multiple cellular events are regulated. Here we report that calcium induces tyrosine phosphorylation in streptolysin O (SLO)-permeabilized human sperm. As expected, pretreatment with tyrphostin A47-a tyrosine kinase inhibitor-abolishes the calcium effect. Interestingly, the calcium-induced increase in tyrosine phosphorylation has a functional correlate in sperm exocytosis. Masking of phosphotyrosyl groups with a specific antibody or inhibition of tyrosine kinases with genistein, tyrphostin A47, and tyrphostin A51 prevent the acrosome reaction. By reversibly sequestering intra-acrosomal calcium with a photo-inhibitable chelator, we show a requirement for protein tyrosine phosphorylation late in the exocytotic pathway, after the efflux of intra-acrosomal calcium. Both mouse and human sperm contain highly active tyrosine phosphatases. Importantly, this activity declines when sperm are incubated under capacitating conditions. Inhibition of tyrosine phosphatases with pervanadate, bis(N,N-dimethylhydroxoamido)hydroxovanadate, ethyl-3,4-dephostatin, and phenylarsine oxide prevents the acrosome reaction. Our results show that both tyrosine kinases and phosphatases play a central role in sperm exocytosis.     

Differences in the mechanism for high- versus moderate-density fibroblast-populated collagen lattice contraction

The free-floating fibroblast-populated collagen lattice (FPCL) model introduced by Bell contains 0.5 x 10(5) cell/ml and here is defined as a moderate-density FPCL (MD-FPCL). One modification of the model is to increase the cell density by a factor of 10, where 5 x 10(5) cells/ml defines a high-density FPCL (HD-FPCL). The initial detection of HD-FPCL contraction is 2 h, whereas MD-FPCL is later, 6 h. A contracted HD-FPCL has a doughnut-like appearance, due to the high density of cells accumulating at the periphery. A contracted MD-FPCL is a flattened disc. The compacted collagen of MD-FPCL lattice exhibits a strong birefringence pattern due to organized collagen fiber bundles. In contracted HD-FPCL, a minimal birefringence develops, indicating minimal organization of collagen fiber bundles. MD-FPCL contraction was reduced with less than 10% serum; the disruption of microtubules, uncoupling of gap junctions, inhibition of tyrosine kinases, and addition of a blocking antibody to alpha2beta1 collagen integrin. Making HD-FPCL with only 1% serum or including the inhibitory agents had only minimal affect on lattice contraction. On the other hand, platelet-derived growth factor stimulated HD-FPCL contraction but had no influence on MD-FPCL contraction. It is suggested that the mechanism for HD-FPCL contraction is limited to the process of cells spreading. HD-FPCL contraction is independent of collagen organization, microtubules, gap junctions, alpha2beta1 integrin, and tyrosine phosphorylation. MD-FPCL contraction involves collagen organization and is optimized by the involvement of microtubules, gap junctions, alpha2beta1 integrin, and tyrosine phosphorylation. When studying cell physiology in a collagen matrix, cell-density influences need to be considered.     

Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells

Previous studies demonstrated that ICAM-1 ligation on human pulmonary microvascular endothelial cells (ECs) sequentially induces activation of xanthine oxidase and p38 MAPK. Inhibition of these signaling events reduces neutrophil migration to the EC borders. This study examined the role of SRC tyrosine kinases in ICAM-1-initiated signaling within these ECs. Cross-linking ICAM-1 on tumor necrosis factor-alpha-pretreated ECs induced an increase in the activity of SRC tyrosine kinases. This increase was inhibited by allopurinol (a xanthine oxidase inhibitor), Me2SO (a hydroxyl radical scavenger), or deferoxamine (an iron chelator). Phenylarsine oxide, a tyrosine phosphatase inhibitor, reduced the base-line activity of SRC as well as the increase in SRC activity induced by ICAM-1 cross-linking. Specific inhibition of the protein expression of the SRC homology 2-containing protein-tyrosine phosphatase-2 (SHP-2) by an antisense oligonucleotide prevented the induced SRC activation but had no effect on the basal SRC activity. Activation of SRC tyrosine kinases was accompanied by tyrosine phosphorylation of ezrin at Tyr-146, which was inhibited by PP2, an SRC tyrosine kinase inhibitor. Moreover, PP2 completely inhibited p38 activation, suggesting a role for SRC tyrosine kinases in p38 activation. These data demonstrate that ICAM-1 ligation activates SRC tyrosine kinases and that this activation requires SHP-2 as well as production of reactive oxygen species generated from xanthine oxidase. Activation of SRC tyrosine kinases in turn leads to tyrosine phosphorylation of ezrin, as well as activation of p38, a kinase previously identified to be required for cytoskeletal changes induced by ICAM-1 ligation and for neutrophil migration along the EC surface.     

Regulation of TRPM8 channel activity by Src-mediated tyrosine phosphorylation

The transient receptor potential melastatin type 8 (TRPM8) receptor channel is expressed in primary afferent neurons where it is the main transducer of innocuous cold temperatures and also in a variety of tumors, where it is involved in progression and metastasis. Modulation of this channel by intracellular signaling pathways has therefore important clinical implications. We investigated the modulation of recombinant and natively expressed TRPM8 by the Src kinase, which is known to be involved in cancer pathophysiology and inflammation. Human TRPM8 expressed in HEK293T cells is constitutively tyrosine phosphorylated by Src which is expressed either heterologously or endogenously. Src action on TRPM8 potentiates its activity, as treatment with PP2, a selective Src kinase inhibitor, reduces both TRPM8 tyrosine phosphorylation and cold-induced channel activation. RNA interference directed against the Src kinase diminished the extent of PP2-induced functional downregulation of TRPM8, confirming that PP2 acts mainly through Src inhibition. Finally, the effect of PP2 on TRPM8 cold activation was reproduced in cultured rat dorsal root ganglion neurons, and this action was antagonized by the protein tyrosine phosphatase inhibitor pervanadate, confirming that TRPM8 activity is sensitive to the cellular balance between tyrosine kinases and phosphatases. This positive modulation of TRPM8 by Src kinase may be relevant for inflammatory pain and cancer signaling.     

The functional synergy between IL-12 and IL-2 involves p38 mitogen-activated protein kinase and is associated with the augmentation of STAT serine phosphorylation

IL-12 and IL-2 can stimulate mitogen- or CD3-activated T cells to proliferate, produce IFN-gamma, and kill tumor cells. The magnitude of these functional responses is greatly augmented when T cells are activated by the combination of IL-12 and IL-2. Although peripheral blood T cells are largely unresponsive to these cytokines without prior activation, a small subset of CD8+ T cells (CD8+CD18bright) is strongly activated by the combination of IL-12 and IL-2. In this report we show that the functional synergy between IL-12 and IL-2 in CD8+CD18bright T cells correlates with the activation of the stress kinases, p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase (SAPK)/Jun N-terminal kinase, but not with the activation of the extracellular signal-regulated kinases. The functional synergy between IL-2 and IL-12 is also associated with a prominent increase in STAT1 and STAT3 serine phosphorylation over that observed with IL-12 or IL-2 alone. By contrast, STAT tyrosine phosphorylation is not augmented over that seen with either cytokine alone. A specific inhibitor of p38 MAP kinase completely inhibits the serine phosphorylation of STAT1 and STAT3 induced by IL-12 and IL-2 and abrogates the functional synergy between IL-12 and IL-2 without affecting STAT tyrosine phosphorylation. This suggests that p38 MAP kinase may play an important role in regulating STAT serine phosphorylation in response to the combination of IL-12 and IL-2. Furthermore, these findings indicate that the optimal activation of T cells by IL-12 and IL-2 may depend on an interaction between the p38 MAP kinase and Janus kinase/STAT signaling pathways.     

Cdk-inhibitory activity and stability of p27Kip1 are directly regulated by oncogenic tyrosine kinases

p27Kip1 controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks). Here we show that Cdk inhibition and p27 stability are regulated through direct phosphorylation by tyrosine kinases. A conserved tyrosine residue (Y88) in the Cdk-binding domain of p27 can be phosphorylated by the Src-family kinase Lyn and the oncogene product BCR-ABL. Y88 phosphorylation does not prevent p27 binding to cyclin A/Cdk2. Instead, it causes phosphorylated Y88 and the entire inhibitory 3(10)-helix of p27 to be ejected from the Cdk2 active site, thus restoring partial Cdk activity. Importantly, this allows Y88-phosphorylated p27 to be efficiently phosphorylated on threonine 187 by Cdk2 which in turn promotes its SCF-Skp2-dependent degradation. This direct link between transforming tyrosine kinases and p27 may provide an explanation for Cdk kinase activities observed in p27 complexes and for premature p27 elimination in cells that have been transformed by activated tyrosine kinases.     

Critical role for hematopoietic cell kinase (Hck)-mediated phosphorylation of Gab1 and Gab2 docking proteins in interleukin 6-induced proliferation and survival of multiple myeloma cells

Interleukin-6 (LI-6) is a known growth and survival factor in multiple myeloma via activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling cascade. In this report we show that Grb2-associated binder (Gab) family adapter proteins Gab1 and Gab2 are expressed by multiple myeloma cells; and that interleukin-6 induces their tyrosine phosphorylation and association with downstream signaling molecules. We further demonstrate that these events are Src family tyrosine kinase-dependent and specifically identify the role of hematopoietic cell kinase (Hck) as a new Gab family adapter protein kinase. Conversely, inhibition of Src family tyrosine kinases by the pyrazolopyrimidine PP2, as in kinase-inactive Hck mutants, significantly reduces IL-6-triggered activation of extracellular signal-regulated kinase and AKT-1, leading to significant reduction of multiple myeloma cell proliferation and survival. Taken together, these results delineate a key role for Hck-mediated phosphorylation of Gab1 and Gab2 docking proteins in IL-6-induced proliferation and survival of multiple myeloma cells and identify tyrosine kinases and downstream adapter proteins as potential new therapeutic targets in multiple myeloma.     

Tyrosine kinase inhibition affects type 1 angiotensin II receptor internalization.

Growth factor receptors activate tyrosine kinases and undergo endocytosis. Recent data suggest that tyrosine kinase inhibition can affect growth factor receptor internalization. The type 1 angiotensin II receptor (AT1R) which is a G-protein-coupled receptor, also activates tyrosine kinases and undergoes endocytosis. Thus, we examined whether tyrosine kinase inhibition affected AT1R internalization. To verify protein tyrosine phosphorylation, both LLCPKCl4 cells expressing rabbit AT1R (LLCPKAT1R) and cultured rat mesangial cells (MSC) were treated with angiotensin II (Ang II) [1-100 nM] then solubilized and immunoprecipitated with antiphosphotyrosine antisera. Immunoblots of these samples demonstrated that Ang II stimulated protein tyrosine phosphorylation in both cell types. Losartan [1 microM], an AT1R antagonist, inhibited Ang II-stimulated protein tyrosine phosphorylation. LLCPKAT1R cells displayed specific 125I-Ang II binding at apical (AP) and basolateral (BL) membranes, and both AP and BL AT1R activated tyrosine phosphorylation. LLCPKAT1R cells, incubated with genistein (Gen) [200 microM] or tyrphostin B-48 (TB-48) [50 microM], were assayed for acid-resistant specific 125I-Ang II binding, a measure of Ang II internalization. Both Gen (n = 7) and TB-48 (n = 3) inhibited AP 125I-Ang II internalization (80+/-7% inhibition; p<0.025 vs. control). Neither compound affected BL internalization. TB-1, a non-tyrosine kinase-inhibiting tyrphostin, did not affect AP 125I-Ang II endocytosis (n = 3), suggesting that the TB-48 effect was specific for tyrosine kinase inhibition. Incubating MSC with Gen (n = 5) or herbimycin A [150 ng/ml] (n = 4) also inhibited MSC 125I-Ang II internalization (82+/-11% inhibition; p<0.005 vs. control). Thus, tyrosine kinase inhibition prevented Ang II internalization in MSC and selectively decreased AP Ang II internalization in LLCPKAT1R cells suggesting that AP AT1R in LLCPKAT1R cells and MSC AT1R have similar endocytic phenotypes, and tyrosine kinase activity may play a role in AT1R internalization.