• BMB Reports
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• 제34권1호
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• pp.33-38
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• 2001

The human PTK6 (also known as Brk) polypeptide, which is deduced from its full-length cDNA, represents a non-receptor protein tyrosine kinase (PTK). It contains SH3, SH2, and tyrosine kinase catalytic domains that are closely related to Src family members. We generated an antihuman PTK6 antibody by immunizing rabbits with a PTK6-specific oligopeptide conjugated to BSA, which corresponds to 11 amino acid residues near the C-terminus. An immunoblot analysis with the antibody detected an expected 52-kDa band in various mammalian transformed cell lines. Immunoprecipitation and immunoblot analyses demonstrated that PTK6 is phosphorylated on the tyrosine residues) and interacts with approximately a 23-kDa tyrosine-phosphorylated polypeptide (most likely a substrate of PTK6) in breast carcinoma T-47D cells. An immunofluorescence analysis demonstrated that PTK6 is localized throughout the cytoplasm of T-47D cells. These results support a possible role for PTK6 in the intracellular signal transduction through tyrosine phosphorylation.

• Journal of Applied Biological Chemistry
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• 제48권3호
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• pp.120-126
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• 2005

Endothelial nitric oxide synthase (eNOS) plays a critical role in vascular biology and pathophysiology. Its activity is regulated by multiple mechanisms such as calcium/calmodulin, protein-protein interactions, sub-cellular locations and phosphorylation at various sites. Phosphorylation of eNOS-Ser1177 (based on mouse sequence) has been identified as an important mechanism of eNOS activation. However, signaling pathway leading to it phosphorylation remains controversial. The regulation of eNOS-Ser1177 phosphorylation by Src and protein kinase A (PKA) was investigated in the present study using cultured mouse aorta endothelial cells. Expression of a constitutively active Src mutant in the cells enhanced phosphorylation of eNOS and protein kinase B (Akt). The Src-stimulated phosphorylation was not attenuated by the expression of a dominant negative PKA regulatory subunit. Neither activation nor inhibition of PKA activity had any significant effect on tyrosine phosphorylation of activation or inactivation site in Src. Based on the results of this study, it is suggested that Src/Akt pathway and PKA signaling may regulate eNOS phosphorylation independently. The existence of multiple mechanisms for eNOS phosphorylation may guarantee endothelial nitric oxide production in various cellular contexts which is essential for maintenance of vascular health.

• BMB Reports
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• 제29권1호
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• pp.11-16
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• 1996

The present study showed that receptor-mediated activation of rabbit kidney proximal tubule cells by angiotensin II, the $Ca^{2+}$ ionophore A23187, or the protein kinase C activator phorbol myristate acetate (PMA) all stimulated phospholipase D (PLD). This was demonstrated by the increased formation of phosphatidic acid, and in the presence of 0.5% ethanol, phosphatidylethanol (PEt) accumulation. Angiotensin II leads to a rapid increase in phosphatidic acid and diacylglycerol, and phosphatidic acid formation preceeded the formation of diacylglycerol. This result suggests that some phosphatidic acid seems to be formed directly from phosphatidylcholine hydrolyzed by Pill. On the other hand, EGTA substantially attenuated angiotensin II and A23187-induced PEt formation, and when the cells were pretreated with verapamil angiotensin II-induced Pill activation was completely abolished. These results provide the evidence that calcium ion influx is essential for the agonist-induced Pill activation. In addition, staurosporine, an inhibitor of protein kinase C, strongly inhibited PMA-induced PEt formation, but was ineffective on angiotensin II-induced PEt accumulation. $GTP{\gamma}S$ also stimulates PEt formation in digitonin-permeabilized cells, but pretreatment of the cells with pertussis toxin failed to suppress angiotensin II-induced PEt formation. From these results, we conclude that in the rabbit kidney proximal tubule cells the mechanisms of angiotensin II- and PMA-induced Pill activation are different from each other and mediated via a pertussis toxin-insensitive trimeric G protein.

• BMB Reports
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• 제38권4호
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• pp.457-467
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• 2005

Open reading frame (ORF) 3 on the Choristoneura fumiferana granulovirus (ChfuGV), located in the 11 kb fragment of the BamHI genomic bank encodes a predicted 32-kDa putative kinase protein. Bioinformatics analysis on the predicted amino acid sequence of ChfuGV PK-1 revealed the existence of 11 catalytic subdomains. Sequence analysis within the 5'-untranslated region (5'-UTR) of ChfuGV pk-1 indicates the presence of both putative early and late promoter motifs, indicating that pk-1 may be expressed throughout the infection cycle. Promoter sequence analysis reveals that pk-1 is deprived of a TATA box and appears instead to be regulated by other cis-acting transcriptional regulatory elements. Temporal transcription analysis by RT-PCR confirms the appearance of transcripts detected from 2 h p.i. until 72 h p.i. Northern blot hybridization characterizes pk-1 transcription as a 1.2 kb transcript. Homology comparisons reveal that ChfuGV PK-1 protein is most closely related to Phthorimaea operculalla granulovirus (PoGV) with 80% amino acid identity.

• IMMUNE NETWORK
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• 제21권2호
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• pp.16.1-16.8
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• 2021

Patients with severe coronavirus disease 2019 (COVID-19) demonstrate dysregulated immune responses including exacerbated neutrophil functions. Massive neutrophil infiltrations accompanying neutrophil extracellular trap (NET) formations are also observed in patients with severe COVID-19. However, the mechanism underlying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced NET formation has not yet been elucidated. Here we show that 2 viral proteins encoded by SARS-CoV-2, the nucleocapsid protein and the whole spike protein, induce NET formation from neutrophils. NET formation was ROSindependent and was completely inhibited by the spleen tyrosine kinase inhibition. The inhibition of p38 MAPK, protein kinase C, and JNK signaling pathways also inhibited viral protein-induced NET formation. Our findings demonstrate one method by which SARSCoV-2 evades innate immunity and provide a potential target for therapeutics to treat patients with severe COVID-19.

• Asian-Australasian Journal of Animal Sciences
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• 제29권5호
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• pp.731-738
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• 2016

Glucagon-like peptide-2 (GLP-2) is important for intestinal barrier function and regulation of tight junction (TJ) proteins, but the intracellular mechanisms of action remain undefined. The purpose of this research was to determine the protective effect of GLP-2 mediated TJ and transepithelial electrical resistance (TER) in lipopolysaccharide (LPS) stressed IPEC-J2 cells and to test the hypothesis that GLP-2 regulate TJ and TER through the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway in IPEC-J2 cells. Wortmannin and LY294002 are specific inhibitors of PI3K. The results showed that $100{\mu}g/mL$ LPS stress decreased TER and TJ proteins occludin, claudin-1 and zonula occludens protein 1 (ZO-1) mRNA, proteins expressions (p<0.01) respectively. GLP-2 (100 nmol/L) promote TER and TJ proteins occludin, claudin-1, and zo-1 mRNA, proteins expressions in LPS stressed and normal IPEC-J2 cells (p<0.01) respectively. In normal cells, both wortmannin and LY294002, PI3K inhibitors, prevented the mRNA and protein expressions of Akt and mTOR increase induced by GLP-2 (p<0.01) following with the significant decreasing of occludin, claudin-1, ZO-1 mRNA and proteins expressions and TER (p<0.01). In conclusion, these results indicated that GLP-2 can promote TJ's expression and TER in LPS stressed and normal IPEC-J2 cells and GLP-2 could regulate TJ and TER through the PI3K/Akt/mTOR pathway.

• BMB Reports
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• 제44권10호
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• pp.659-664
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• 2011

As part of the search for biologically active anti-osteoporotic agents that enhance differentiation and mineralization of osteoblastic MC3T3-E1 cells, we identified the ginsenoside Rh2(S), which is an active component in ginseng. Rh2(S) stimulates osteoblastic differentiation and mineralization, as manifested by the up-regulation of differentiation markers (alkaline phosphatase and osteogenic genes) and Alizarin Red staining, respectively. Rh2(S) activates p38 mitogen-activated protein kinase (MAPK) in time- and concentration-dependent manners, and Rh2(S)-induced differentiation and mineralization of osteoblastic cells were totally inhibited in the presence of the p38 MAPK inhibitor, SB203580. In addition, pretreatment with Go6976, a protein kinase D (PKD) inhibitor, significantly reversed the Rh2(S)-induced p38 MAPK activation, indicating that PKD might be an upstream kinase for p38 MAPK in MC3T3-E1 cells. Taken together, these results suggest that Rh2(S) induces the differentiation and mineralization of MC3T3-E1 cells through activation of PKD/p38 MAPK signaling pathways, and these findings provide a molecular basis for the osteogenic effect of Rh2(S).

• 한국발생생물학회지:발생과생식
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• 제4권1호
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• pp.29-35
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• 2000

A phosphatidylinositol 3-kinase (PI3K)는 인슐린 신호전달의 상위구성 요소로 다양한 세포에서 단백질합성을 촉진한다. PI3K와 하위의 mammalian target of rapamycin (mTOR)가 착상전 생쥐 배아의 인슐린 신호전달에 관여하고 있는지의 여부를 조사하고자 하였다. 생쥐의 8-세포기 배아를 인슐린 또는 PI3K및 mTOR의 억제제를 포함한 조건에서 배양하면서 발생율, 할구수, 단백질합성 및 인산화를 조사하였다. 인슐린의 첨가는 포배형성과 부화 등 형태발생을 촉진하며 포배내 평균 할구수, 8-세포기 배아의 단백질 합성과 인산화를 유의하게 증가시켰다. PI3K의 억제제인 wortmannin과 mTOR를 억제하는 rapamycin은 인슐린에 의한 발생율, 포배내, 할구수, 단백질합성의 증가 효과를 상쇄하였다. 오토라디오그라피에서 두종의 인산화단백질인 pp22와 pp30의 인산화가 인슐린 처리에 의해 증가함을 확인하였다. 이상의 결과에서 생쥐 8-세포기 배아의 발생을 촉진하는 인슬린 신호의 전달에 PI3K와 mTOR가 관여함을 알 수 있다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권6호
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• pp.2495-2499
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• 2015

Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 3 (MEKK3) is an important serine/threonine protein kinase and a member of the MAPK family. MEKK3 can effectively activate the MEK/ERK signaling pathway and promote an autocrine growth loop critical for tumor genesis, cell proliferation, terminal differentiation, apoptosis and survival. To explore the relationship between MEKK3 and cell apoptosis, clinicopathology and prognosis, we characterize the expression of MEKK3, pERK and FoxP3 in the renal clear cell carcinoma (RCCC). Protein expression was detected by tissue microarray and immunochemistry in 46 cases of RCCC and 28 control cases. Expression levels of CD3+,CD3+CD4+,CD3+CD8+,CD4+CD25+, CD4+CD25+ FoxP3+ were assessed by flow cytometry and analyzed for their association with pathological factors, correlation and prognosis in RCCC. Expression of MEKK3, pERK and FoxP3 was significantly up-regulated in RCCC as compared to control levels (p<0.01), associated with pathological grade (p<0.05)and clinical stage (p<0.05). CD4+CD25+ Foxp3+ Treg cells were also significantly increased in RCCC patients (p<0.05). Cox multivariate regression analysis showed that MEKK3, pERK expression and patholigical stage were independent prognostic factors in patients with RCCC (p<0.05). MEKK3 can be used as an important marker of early diagnosis and prognostic evaluation in RCCC. It may be associated with imbalance of anti-tumor immunity and overexpression of pERK. Expression of MEKK3 and pERK are significantly increased in RCCC, with protein expression and clinical stage acting as independent prognostic factors.

• 대한의생명과학회지
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• 제12권3호
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• pp.249-254
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• 2006

It has been reported that liver is a very important organ to xenotransplantation. Pig is known to be a most suitable species in transplantation of human organs. However, the physiological function of pig hepatocytes is not clear elucidated. Epidermal growth factor (EGF) is known to be a mitogen in various cell systems. Thus, we examined the effect of EGF on cell proliferation and its related signal cascades in primary cultured pig hepatocytes. EGF stimulates cell proliferation in a dose (>1ng/ml) dependent manner. EGF-induced increase of $[^3H]-thymidine$ incorporation was blocked by AG 1478 ($10^{-6}M$, an EGF receptor antagonist) genistein and herbymycin A (tyrosine kinase inhibitors, $10^{-6}M$), suggesting the role of activation and tyrosine phosphorylation of EGF receptor. In addition, EGF-induced increase of $[^3H]-thymidine$ incorporation was prevented by neomycin $(10^{-4}M)$, U73122 $(10^{-5}M)$ (phospholipase C [PLC] inhibitors), staurosporine ($(10^{-8}M)$, or bisindolylmaleimide I $(10^{-6}M)$ (protein kinase C [PKC] inhibitors), suggesting the role of PLC and PKC. Moreover, EGF-induced increase of $[^3H]-thymidine$ incorporation was blocked by PD 98059 (a p44/42 mitogen activated protein kinase [MAPK] inhibitor), SB 203580 (a p38 MAPK inhibitor), and SP 600125 (a JNK inhibitor). EGF increased the translocation of PKC from cytosol to membrane fraction and activated p42/44 MAPK, p38 MAPK and JNK. In conclusion, EGF stimulates cell proliferation via PKC and MAPK in cultured pig hepatocytes.