Relationship between Tyrosine Phosphorylation and Protein Expression of Insulin Receptor and Insulin Resistance in Gestational Diabetes Mellitus

Summary： The relationship between tyrosine phosphorylation （TP） and protein expression of insulin receptor （InsR） and insulin resistance （IR） in patients with gestational diabetes mellitus （GDM） was investigated. The InsR expression and TP in skeleton muscle tissue were determined by Western blotting and immunoprecipitation in women with GDM （GDM group, n=22）, normal pregnant women （normal pregnancy group, n=22） and normal non-pregnant women （normal non-pregnant group, n=13）. Fasting plasma glucose （FPG） and fasting insulin （FINS） were measured by oxidase assay and immunoradioassay. The results showed that the levels of FPG （5.61±0.78 mmol/L）, FINS （15.42±5.13 mU/L） and Ho- meostasis model assessment-IR （HOMA-IR） （1.21±0.52） in GDM group were significantly higher than those in normal pregnancy group （4.43±0.46 mmol/L, 10.56±3.07 mU/L and 0.80±0.31 respectively） （P〈0.01）. The levels of FINS and HOMA-IR in normal pregnancy group were significantly higher than those in normal non-pregnant group （7.56±2.31 mU/L and 0.47±0.26 respectively） （P〈0.01）. There was no significant difference in the InsR expression level among the three groups （P〉0.05）. TP of InsR with insulin stimulation was significantly decreased in GDM group （0.20±0.05） as compared with normal pregnancy group （0.26±0.06） （P〈0.01）. TP of InsR with insulin stimulation in normal pregnancy group was lower than that in normal non-pregnant group （0.31±0.06） （P〈0.01）. TP of InsR with insulin stimu- lation was negatively related with HOMA-IR in GDM group （r=-0.525, P〈0.01）. There was no correlation between the protein expression of InsR and HOMA-IR in GDM group （r=-0.236, P〉0.05）. It was suggested that there is no significant correlation between the protein expression of InsR in skeletal muscle and IR in GDM, but changes in TP of InsR are associated with IR in GDM.

Crk adaptor protein-induced phosphorylation of Gab1 on tyrosine 307 via Src is important for organization of focal adhesions and enhanced cell migration

Upon growth factor stimulation, the scaffold protein, Gabl, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gabl. We have previously shown that Crk overexpression, which is detectable in various human cancers, induces tyrosine phosphorylation of Gabl without extracellular stimuli. In the present study, the underlying mechanisms were further investigated. Mutational analyses of CrkII demonstrated that the SH2 domain, but not the SH3（N） or the regulatory Y221 residue of CrkII, is critical for the induction of Gabl- Y307 phosphorylation. SH2 mutation of CrkII also decreased the interaction with Gabl. In GST pull-down assay, Crk-SH2 bound to wild-type Gahl, whereas Crk-SH3（N） interacted with the Gabl mutant, which lacks the clus- tered tyrosine region （residues 242-410）. Tyrosine phosphorylation of Gabl was induced by all Crk family proteins, but not other SH2-containing signalling adaptors. Src-family kinase inhibitor, PP2, abrogates Crk-induced tyrosine phosphorylations of Gabl. Y307 phosphorylation was undetectable in fibroblasts lacking Src, Yes, and Fyn, even upon overexpression of Crk, whereas cells lacking only Yes and Fyn still contained Gabl with phosphorylated Y307. Furthermore, Crk induced the phosphorylation of Src-Y416; accordingly the interaction between Crk and Csk was increased. The Gabl-Y307F mutant failed to localize near the plasma membrane even upon HGF stimulation and decreased cell migration. Moreover, Gabl-Y307F disturbed the localization of Crk, FAK, and paxillin, which are the typical components of focal adhesions. Taken together, these results indicate that Crk facilitates tyrosine phosphory- lation of Gabl-Y307 through Src, contributing to the organization of focal adhesions and enhanced cell migration, thereby possibly promoting human cancer development.

Protein tyrosine phosphorylation of the human sperm head during capacitation： immunolocalization and relationship with acquisition of sperm-fertilizing ability

The occurrence of tyrosine phosphorylation （TP） in the sperm head during capacitation has been poorly investigated, and no data exist on the relationship of its dynamics with the acquisition of sperm fertilizing ability. This study localized TP of head proteins in human spermatozoa during capacitation and explored its relationship with acquisition of the ability to display progesterone （P）-stimulated acrosome reactions （ARs） and to penetrate zona-free hamster oocytes. By immunofluorescence, TP immunoreactivity was revealed in the acrosomal region of formaldehyde-fixed/unpermeabilized samples, whereas it was abolished in fixed/permeabilized samples, in which TP immunoreactivity was high in the principal piece. No TP immunoreaetivity was detectable in unfixed spermatozoa. Head TP immunoreactivity was localized externally to the acrosome, close to the cytoplasmic membrane, as assessed by transmission electron microscopy. The increase in head TP was an early event during capacitation, occurring within 1 h in capacitating conditions. At this time, the P-stimulated ARs were also increased, whereas egg penetration was as poor as in uncapacitated spermatozoa. At 5 h of capacitation, the extent of neither head TP nor the P-induced ARs were greater than that at 1 h, whereas egg penetration had significantly increased. Seminal plasma inhibited head TP, P-induced ARs and egg penetration. None of these inhibitory effects, unlike those on tail TP, were prevented by the cAMP analogue dbcAMP （N,2-O-dibutyryladenosine 3＇,5＇-cyclic monophosphate）. In conclusion, head TP is a subsurface event occurring early during capacitation and is closely related to acquisition of the ability to display P-stimulated ARs, whereas the ability to fuse with oolemma and to decondense is a later capacitation-related event.

Tyrosine phosphorylation and bacterial virulence

Protein phosphorylation on tyrosine has emerged as a key device in the control of numerous cellular functions in bacteria.In this article,we review the structure and function of bacterial tyrosine kinases and phosphatases.Phosphorylation is catalyzed by autophosphorylating adenosine triphosphate-dependent enzymes（bacterial tyrosine（BY） kinases） that are characterized by the presence of Walker motifs.The reverse reaction is catalyzed by three classes of enzymes：the eukaryotic-like phosphatases（PTPs） and dual-specific phosphatases;the low molecular weight protein-tyrosine phosphatases（LMW-PTPs）;and the polymerase-histidinol phosphatases（PHP）.Many BY kinases and tyrosine phosphatases can utilize host cell proteins as substrates,thereby contributing to bacterial pathogenicity.Bacterial tyrosine phosphorylation/dephosphorylation is also involved in biofilm formation and community development.The Porphyromonas gingivalis tyrosine phosphatase Ltp1 is involved in a restraint pathway that regulates heterotypic community development with Streptococcus gordonii.Ltp1 is upregulated by contact with S.gordonii and Ltp1 activity controls adhesin expression and levels of the interspecies signal AI-2.

THE IMMUNOREGULATORY EFFECT OF TLSF_(JM) ON THE EXPRESSION OF T CELL IL-2R AND PROTEIN TYROSINE PHOSPHORYLATION

The immunoregulatory effect of TLSFJM on the expression of T cell IL- 2R and protein tyrosine phosphorylation ( PTP ) was investigated by immunohistochemistry technique. The results showed that TLSFJMcan markedly suppress the expression of IL-2R and PTP on PHA or TPA-stimulated human PBMC and murine IL-2 dependent cell line CTLL-2. However, there was no effect of TLSFJMon the production of IL-1, IL-2 and IL-6 that play an important role in the course of T lymphocyte proliferation and differentiation.

Tyrosine phosphorylation of monocyte-derived macrophage proteins in buffalo (Bubalus bubalis): A potential phenotype of natural resistance

The aim of this work was to explore the possibility of using the presence of tyrosine-phosphorylated macrophage proteins as a phenotype of natural resistance. Tyrosine-phosphorylation of macrophage proteins was investigated in 18 buffaloes, that carried either the resistant, or the non-resistant, Natural Resistance-Associated Macrophage Protein one (NRAMP1) genotype, that various authors have associated with susceptibility to intracellular bacterial diseases. Monocyte-derived macrophages were Interferon-gamma (IFN-γ) stimulated and tyrosine-phosphorylation was assessed by Western blotting. Evidence of phosphorylation after IFN-γ stimulation was shown by 75% of the buffaloes carriers of the resistant genotype, and by 20% of the carriers of the non-resistant genotype (Chisquare value between the groups = 5.44;P = 0.02). The study of the Proteoma of monocyte-derived macrophages might open the way to the genetic control of disease resistance.

Lithium decreased NR2B tyrosine phosphorylation and interactions of NR2B and PSD-95 with Src in rat hippocampus following cerebral ischemia

Objective： To study the effects of chronic lithium on N-methyl-d-aspartate receptor subunit 2B （NR2B） tyrosine phosphorylation and the interactions of NR2B and PSD-95 with Src induced by cerebral ischemia/reperfusion （I/R）. Methods： Transient （15min） cerebral ischemia was induced by four-vessel occlusion procedure in SD rats. Immunoprecipitation （IP） and immunoblotting （IB） were performed to investigate the phosphorylation and interactions of proteins. The effects of lithium on tyrosine phosphorylation of NR2B and its interactions with PSD-95 and Src were examined. Results： Transient cerebral ischemia 15 rain followed by reperfusion 6h （I/R 6h） caused a significant increase in tyrosine phosphorylation of NR2B. Administration of LiCI for 7days before ischemia caused a profound decrease in tyrosine phosphorylation of NR2B. Similiarly. the interactions of NR2B and PSD-95 with Src were also enhanced by I/R 6h. moreover, these interactions were also inhibited by chronic lithium. Conclusion： Pretreatment with lithium decrease tyrosine phosphorylation of NR2B and interactions of NR2B and PSD-95 with Src during cerebral I/R.

Tyrosine 23 Phosphorylation of Annexin A2 Promotes Proliferation,Invasion,and Stat3 Phosphorylation in the Nucleus of Human Breast Cancer SK-BR-3 Cells

Objective To investigate the role of tyrosine 23 （Tyr23） phosphorylation of Annexin A2 （Anxa2） in regulating the proliferation and invasion of human breast cancer SK-BR-3 cells. Methods A panel of lentivirus plasmids expressing Anxa2-wide type （Ana2-WT）, Anxa2-Y23A, and Anxa2-Y23D was generated and infected with SK-BR-3 cells. The monoclonal strains were screened. The expression of Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D was determined by Western blot analysis. The ability of the cells to proliferate was detected through an MTT [3-（4,5-Dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide] test. Boyden chamber assays were employed to examine migration and invasion abilities. The interaction between Anxa2 and Stat3 was analyzed by immunoprecipitation analyses. Nucleoprotein and cytosolic protein were extracted from SK-BR-3, Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D cells to analyze the expression and localization of Stat3 phosphorylation. Results The monoclonal strains constitutively expressing Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D were screened. Both Anxa2-WT and Anxa2-Y23D enhanced the proliferation, migration and invasion abilities of SK-BR-3 cells （P〈0.05）. Immunoprecipitation analysis revealed that Anxa2 and Stat3 interacted with each other, and the expression of Stat3 phosphorylation in the nucleus was enhanced by Anxa2-Y23D. Conclusions Tyr23 phosphorylation of Anxa2 promotes the proliferation and invasion of human breast cancer SK-BR-3 cells and the phosphorylation of Stat3 in the nucleus.

Effect of Metabolites of Fusarium Solani on Tyrosine Phosphorylation in Cultivated Cells of Solarium Tuberosum

Elicitor-induced phosphorylation of tyrosine residues in proteins of potato was studied. Proteins of crude extract of suspension culture of potato were analyzed by one- and two-dimensional electrophoresis followed by Western blotting with monoclonal antibodies PY20 to phosphotyrosine proteins. One- and two-dimensional electrophoresis revealed l l and 25 tyrosine-phosphorylated proteins, respectively. Glycoprotein increased the phosphorylation level of most of these proteins.

Overexpression of tyrosine phosphorylated proteins in reproductive tissues of polycystic ovary syndrome rats induced by letrozole

Objective:To identify the alteration of tyrosine phosphorylated protein expression in rats with polycystic ovary syndrome(PCOS).Methods:Sixteen female Sprague-Dawley rats were divided into the control and letrozole-induced PCOS groups.The oestrus cycle of rats was performed by vaginal smear.Sex hormones and morphology of the ovary,oviduct,and uterus were observed.Expressions and intensity of androgen receptor and tyrosine phosphorylated proteins of reproductive organs were investigated by Western blot.Results:Various polycysts and increased androgen receptor expression were present in the ovary of the PCOS group.The levels of follicle-stimulating hormone and testosteone were significantly higher in the PCOS group while progesterone and estradiol levels were significantly decreased as compared with the control group(P<0.05).Only the size of uterus in the PCOS group was significantly smaller than the control group.However,the density of collagen fibers observed in PCOS uterus was greater than the control group.Moreover,tyrosine phosphorylated proteins were significantly overexpressed in ovary(52,42,and 28 kDa),oviduct(72,56,42,and 28 kDa),and uterus(53 and 42 kDa)of the PCOS group compared to the control group.Conclusions:Presence of tyrosine phosphorylated proteins in the ovary,oviduct and uterus suggests that overexpression of tyrosine phosphorylated proteins may be involved in potential mechanism of female infertility especially in PCOS.

Protein tyrosine phosphatase non-receptor type 2 andinflammatory bowel disease

Genome wide association studies have associated single nucleotide polymorphisms within the gene locus encoding protein tyrosine phosphatase non-receptor type 2(PTPN2) with the onset of inflammatory bowel disease(IBD) and other inflammatory disorders. Expression of PTPN2 is enhanced in actively inflamed intestinal tissue featuring a marked up-regulation in intestinal epithelial cells. PTPN2 deficient mice suffer from severe intestinal and systemic inflammation and display aberrant innate and adaptive immune responses. In particular, PTPN2 is involved in the regulation of inflammatory signalling cascades, and critical for protecting intestinal epithelial barrier function, regulating innate and adaptive immune responses, and finally for maintaining intestinal homeostasis. On one hand, dysfunction of PTPN2 has drastic effects on innate host defence mechanisms, including increased secretion of pro-inflammatory cytokines, limited autophagosome formation in response to invading pathogens, and disruption of the intestinal epithelial barrier. On the other hand, PTPN2 function is crucial for controlling adaptive immune functions, by regulating T cell proliferation and differentiation as well as maintaining T cell tolerance. In this way, dysfunction of PTPN2 contributes to the manifestation of IBD. The aim of this review is to present an overview of recent findings on the role of PTPN2 in intestinal homeostasis and the impact of dysfunctional PTPN2 on intestinal inflammation.

Effect of genistein on acrosome reaction and zona pellucida binding independent of protein tyrosine kinase inhibition in bull

Aim： To investigate if the phytoestrogen, genistein, affects essential functions of cryopreserved bovine spermatozoa. Methods： The effect of genistein upon motility was assessed by computer-assisted motion analysis. Hemizona assay was performed to detect the ability of spermatozoa binding to the zona pellucida. The inducibility of the acrosome reaction using progesterone and ZP3-6 peptide was analysed by fluorescein-conjugated Pisum sativum agglutinin （FITC-PSA）/Hoechst 33258 double staining. Capacitation after incubation with genistein was assessed by the chlortetracycline （CTC） assay. Immunoblots showed the pattern of protein tyrosine phosphorylation of cryopreserved bovine spermatozoa. Results： Immunodetection of tyrosine-phosphorylated proteins showed that genistein did not affect tyrosine phosphorylation in cryopreserved bovine spermatozoa. However, genistein significantly reduced the progesterone- and ZP3-6 peptide-mediated induction of the acrosome reaction and led to a dose-dependent inhibition of sperm-zona pellucida binding; while sperm motility and capacitation were not affected by this phytoestrogen, as indicated by computer-assisted sperm motion analysis and the CTC assay, respectively. Conclusion： Our results suggest that in cryopreserved bovine spermatozoa, genistein affects a protein tyrosine phosphorylation-independent signal transduction pathway that is involved in sperm capacitation, the acrosome reaction and sperm-zona pellucida binding.

ADHESION-INDUCE PROTEIN TYROSINE PHOSPHORY-LATION IS ASSOCIATED WITH INVASIVE AND METASTATIC POTENTIALS IN B16-BL6 MELANOMA CELLS

Objective: The interaction of cancer cell with extracellular matrix (ECM) happens as an earlier and specific event in the invasive and metastatic cascade. To explore the key element(s) in cancer metastasis and observe the cellECM interaction and its role. Methods: To interrupt the cellECM interaction by suppression of adhesioninduced protein tyrosine phosphorylation with protein tyrosine kinase inhibitor genistein in B16B16 mouse melanoma cells. Results: When B16BL6 cells attached to Matrigel, a solubilized basement membrane preparation from EHS sarcoma, a 125 kDa protein increased its phosphotyrosine content dramatically. In contrast, when the cells were pretreated with 20μM or 30 μM genistein for 3 days, it was revealed a less increase in the phosphotyrosine content of this 125 kDa protein in response to cell attachment to ECM was revealed with immunoblot analysis. Accompanied by the lower level of adhesioninduced protein tyrosine phosphorylation the genisteintreated cells exhibited a decrease in their capabilities of adhesion to Matrigel and invasion through reconstituted basement membrane. The potentials of and forming lung metastatic nodules were also shown to be decreased dramatically in these genisteintreated cells. Conclusion: It was suggested that protein tyrosine phosphorylation in cellECM interaction might be associated with invasive and metastatic potentials in cancer cells.

EphA3 functions are regulated by collaborating phosphotyrosine residues

Ephrin ligands interact with Eph receptors to regulate a wide variety of biological and pathological processes. Recent studies have identified several downstream pathways that mediate the functions of these receptors. Activation of the receptors by ephrin binding results in the phosphorylation of the receptor tyrosine residues. These phospho- rylated residues serve as docking sites for many of the downstream signaling pathways. However, the relative contributions of different phosphotyrosine residues remain undefined. In the present study, we mutated each individual tyrosine residues in the cytoplasmic domain of EphA3 receptor and studied the effects using cell migration, process retraction, and growth cone collapse assays. Stimulation of the EphA3 receptor with ephrin-A5 inhibits 293A cell mi- gration, reduces NG108-15 cell neurite outgrowth, and induces growth cone collapse in hippocampal neurons. Muta- tion of either Y602 or Y779 alone partially decreases EphA3-induced responses. Full abrogation can only be achieved with mutations of both Y602 and Y779. These observations suggest a collaborative model of different downstream pathways.

Regulatory role of protein tyrosine phosphorylation in platelet activating factor-induced signal transduction in platelets

目的：研究蛋白酪氨酸磷酸化（ＰＴＰ）在血小板激活因子（ＰＡＦ）诱导血小板信号传导中的作用．方法：用水洗兔血小板考查Ｇｅｎ抑制聚集及５羟色胺释放，Ｆｕｒａ２和ＢＣＥＣＦ负载测胞内钙及ｐＨ，特异性抗酪氨酸单抗及免疫印迹法检测ＰＴＰ．结果：Ｇｅｎ１００和２００μｍｏｌ·Ｌ－１分别抑制ＰＡＦ诱导的５羟色胺释放为２３７％±２０％及４１％±８％，对胞内钙增加和Ｎａ＋／Ｈ＋交换也有抑制作用．ＰＡＦ增加Ｍｒ为７００００，６００００，５００００，４２０００／４００００，３４０００的ＰＴＰ．Ｇｅｎ２００，４００μｍｏｌ·Ｌ－１明显抑制该效应．用Ｓｔａ２０ｎｍｏｌ·Ｌ－１，ＢＡＰＴＡ２００μｍｏｌ·Ｌ－１，依他酸２ｍｍｏｌ·Ｌ－１，分别阻断ＰＫＣ及胞内钙增加和内流，也减少ＰＴＰ形成．结论：ＰＴＰ参与ＰＡＦ诱导血小板信号传导途径，ＰＫＣ活化和胞内钙动员对ＰＴＰ有调节作用．

Effect of nitric oxide-induced tyrosine phosphorylation of calcium-activated potassium channel α subunit on vascular hyporesponsiveness in rats

Objective: To study the effect of nitric oxide-induced tyrosine phosphorylation of large-conductance calcium-activated potassium (BK Ca) channel α subunit on vascular hyporesponsiveness in rats. Methods: A total of 46 Wistar rats of either sex, weighing 250 g±20 g, were used in this study. Models of vascular hyporesponsiveness induced by hemorrhagic shock (30 mm Hg for 2 hours) in vivo and by L-arginine in vitro were established respectively. The vascular responsiveness of isolated superior mesenteric arteries to norepinephrine was observed. Tyrosine phosphorylation of BK Ca α subunit was evaluated with methods of immunoprecipitation and Western blotting. Results: In the smooth muscle cells of the superior mesenteric arteries, the expression of BK Ca α subunit tyrosine phosphorylation increased following hemorrhagic shock, and L-arginine could induce BK Ca channel α subunit tyrosine phosphorylation in a time- and dose-dependent manner. L-NAME (Nω-nitro-L-arginine-methyl-ester), a nitric oxide synthetase inhibitor, could partly restore the decreased vasoresponsiveness of the superior mesenteric arteries after hemorrhagic shock in rats. Down-regulating the protein tyrosine phosphorylation with genistein, a widely-used special protein tyrosine kinase inhibitor, could partly improve the decreased vasoresponsiveness of the superior mesenteric arteries induced by L-arginine in vitro, while up-regulating the protein tyrosine phosphorylation with Na3VO4, a protein tyrosine phosphatase inhibitor, could further decrease the nitric oxide-induced vascular hyporesponsiveness, which could be partly ameliorated by 0.1 mmol/L tetrabutylammonium chloride (TEA), a selective BK Ca inhibitor at this concentration. Conclusions: Nitric oxide can induce the tyrosine phosphorylation of BK Ca α subunit, which influences the vascular hyporesponsiveness in hemorrhagic shock rats or induced by L-arginine in vitro.

Synaptic non-GluN2B-containing NMDA receptors regulate tyrosine phosphorylation of GluN2B 1472 tyrosine site in rat brain slices

Activation of N-methyl-D-aspartate receptors（NMDARs）mediates changes in the phosphorylation status of the glutamate receptors themselves.Previous studies have indicated that during synaptic activity,tyrosine kinases（Src and Fyn）or phosphatases（PTPαand STEP）are involved in regulating the phosphorylation of NMDARs.In this study,we used immunoblotting to investigate the role of an NMDAR subpopulation on the phosphorylation level of the GluN2B subunit at the Y1336 and Y1472sites in rat brain slices after NMDA treatment.We found that NMDA stimulation dramatically decreased the phosphorylation level of GluN2B at Y1472 in a dose-and time-dependent manner,but not at Y1336.Extrasynaptic NMDAR activation did not reduce the phosphorylation of GluN2B at Y1472.In addition,ifenprodil,a selective antagonist of GluN2Bcontaining NMDARs,did not abolish the decreased phosphorylation of GluN2B at Y1472 triggered by NMDA.These results suggest that the activation of synaptic GluN2A-containing NMDARs is required for the decreased phosphorylation of GluN2B at Y1472that is induced by NMDA treatment in rat brain slices.

Src mediatesβ-adrenergic receptor induced YAP tyrosine phosphorylation

The Hippo pathway is a newly identified pathway and evolutionarily conserved from flies to humans mainly regulating cell proliferation.Transcriptional co-activator Yes-associated protein(YAP)functions as a major downstream effector and key node of the Hippo pathway.Phosphorylation of YAP is critical to regulate YAP activity and its corresponding functions.β-adrenergic receptor(β-AR),a typical G protein coupled receptor(GPCR),mediates proliferation in various cell types and regulates multiple physical and pathological processes.However,the role ofβ-AR in regulating YAP remains elusive.Here,we report thatβ-AR can obviously stimulate YAP tyrosine phosphorylation.The mechanism is thatβ-AR stimulation results in tyrosine kinase Src activation and Src phosphorylates YAP tyrosine at Y357.Further studies demonstrate that inhibition of Src kinase activity can obviously alleviateβ-AR induced YAP tyrosine phosphorylation and cell proliferation.We conclude thatβ-AR can induce YAP tyrosine phosphorylation and also establish the Src/YAP pathway as a critical signaling branch downstream of GPCR.

Cytoskeleton-associated protein tyrosine phosphorylation involved in induction of differentiation in mouse melanoma cells

The malignancy of a cancer is due partly to its poor differentiation. Genistein, a protein tyrosine kinase inhibitor, is found to induce the highly malignant B16-BL6 mouse melanoma cells to differentiate to mature phenotypes. When Triton X-100 insoluble fraction of the differentiated cells is prepared and analyzed, tyrosine phosphorylation levels of three cytoskeleton-associated proteins (65, 60 and 53 ku respectively) are found to decrease dramatically. But no any change is found when phosphotyrosine contents of the cytosol fraction or the total cellular protein preparations are evaluated. It is concluded that cytoskeleton-associated protein tyrosine phosphorylation may be involved in the control of differentiation of cancer cells. The decrease of phosphotyrosine contents of cytoskeleton-associated proteins may be one of the important mechanisms underlying the differentiation induction of cancer cells by anticancer agents.

Label-Free,Versatile,Real-Time,and High-Throughput Monitoring of Tyrosine Phosphorylation Based on Reversible Configuration Freeze

Tyrosine Phosphorylation(pTyr)is a critical and ubiquitous regulation mechanism in biology that plays a central role in controlling intracellular signaling networks.Precise recognition and specific detection of pTyr peptides have been of great importance for both discoveries of disease biomarkers and screening of therapeutic drugs,especially cancers.Here we report a label-free,versatile,realtime,and high-throughput detection strategy for phosphopeptide(PP)based on reversible configuration freeze of a unique hemicyanine-labeled 2-(2′-hydroxyphenyl)-4-methyloxazole(H-HPMO).By taking advantage of the“OFF–ON”transition of fluorescence,H-HPMO–Cu^(2+)complex displays a highly sensitive and selective response to PPs with modified sites on serine,threonine,and tyrosine.Specific recognition of Tyr PPs is achieved by performing a simple logic gate operation and introducing Ca^(2+)interference as an input.This PP detection approach is universal for various peptide sequences and displays high potential in large-scale kinase inhibitor screening,which will promote the development of targeted anticancer drugs.