C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK)—endogenous negative regulators of Src-family protein kinases

C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK) are endogenous inhibitors of the Src-family protein tyrosine kinases (SFKs). Since constitutive activation of SFKs contributes to cancer formation and progression, to prevent excessive activation of SFKs, their activity in normal cells is kept at the basal level by CSK and CHK. CSK and CHK inactivate SFKs by specifically phosphorylating a consensus tyrosine (called Y_T) near their C-termini. Upon phosphorylation, the phospho-Y_T engages in intramolecular interactions that lock the SFK molecule in an inactive conformation. SFKs are anchored to the plasma membrane, while CSK and CHK are localized predominantly in the cytosol. To inhibit SFKs, CSK and CHK need to translocate to the plasma membrane. Recruitment of CSK and CHK to the plasma membrane is mediated by the binding of their SH2, SH3 and/or kinase domains to specific transmembrane proteins, G-proteins and adaptor proteins located near the plasma membrane. For CSK, membrane recruitment often accompanies activation. CSK and CHK employ two types of direct interactions with SFKs to achieve efficient Y_T phosphorylation: (i) short-range interactions involving binding of the active sites of CSK and CHK to specific residues near Y_T, (ii) long-range non-catalytic interactions involving binding of SFKs to motifs located distally from the active sites of CSK and CHK. The interactions between CSK and SFKs are transient in nature. Unlike CSK, CHK binds tightly to SFKs to form stable protein complexes. The binding is non-catalytic as it is independent of Y_T. More importantly, the tight binding alone is sufficient to completely inhibit SFKs. This non-catalytic inhibitory binding represents a novel mechanism employed by CHK to inhibit SFKs. Given that SFKs are implicated in cancer development, compounds mimicking the non-catalytic inhibitory mechanism of CHK are potential anti-cancer therapeutics.     

C-reactive protein promotes atherosclerosis by increasing LDL transcytosis across endothelial cells

Background and Purpose

The retention of plasma low-density lipoprotein (LDL) particles in subendothelial space following transcytosis across the endothelium is the initial step of atherosclerosis. Whether or not C-reactive protein (CRP) can directly affect the transcytosis of LDL is not clear. Here we have examined the effect of CRP on transcytosis of LDL across endothelial cells and have explored the underlying mechanisms.

Experimental Approach

Effects of CRP on transcytosis of FITC-labelled LDL were examined with human umbilical vein endothelial cells and venous rings in vitro and, in vivo, ApoE^-/- mice. Laser scanning confocal microscopy, immunohistochemistry and Oil Red O staining were used to assay LDL.

Key Results

CRP increased transcytosis of LDL. An NADPH oxidase inhibitor, diphenylene iodonium, and the reducing agent, dithiothreitol partly or completely blocked CRP-stimulated increase of LDL transcytosis. The PKC inhibitor, bisindolylmaleimide I and the Src kinase inhibitor, PP2, blocked the trafficking of the molecules responsible for transcytosis. Confocal imaging analysis revealed that CRP stimulated LDL uptake by endothelial cells and vessel walls. In ApoE^-/- mice, CRP significantly promoted early changes of atherosclerosis, which were blocked by inhibitors of transcytosis.

Conclusions and Implications

CRP promoted atherosclerosis by directly increasing the transcytosis of LDL across endothelial cells and increasing LDL retention in vascular walls. These actions of CRP were associated with generation of reactive oxygen species, activation of PKC and Src, and translocation of caveolar or soluble forms of the N-ethylmaleimide-sensitive factor attachment protein.     

氯胺酮拮抗脑缺血诱导大鼠海马脑区c-Src磷酸化及其与Pyk2的结合

目的 :研究脑缺血损伤诱导非受体酪氨酸蛋白激酶Src之丝、苏、酪氨酸残基磷酸化及其与Pyk2结合的变化并探讨其可能的调节机制。方法 :四动脉结扎模型诱导大鼠前脑缺血损伤 ,免疫沉淀和免疫印迹法观察Src氨基酸残基磷酸化及其与Pyk2结合的变化。 结果 :缺血 15min后复灌 ,Src的Tyr 4 16和Ser而不是Thr的磷酸化有明显增加 ,且复灌 6hTyr 4 16磷酸化升至最高 ,而Ser磷酸化 1h升至顶峰 ,它们分别是正常组的 2 .8和 2 .3倍 ;此外 ,缺血 /复灌明显诱导Src与Pyk2的结合 ,相似于Tyr 4 16磷酸化变化 ,复灌 6h升至最高。 5 0mg/kg的氯胺酮能明显抑制缺血 /复灌诱导的SrcTyr 4 16磷酸化及其与Pyk2结合增加 ,与对照组比有显著性差异 (P <0 .0 5 )。结论 :脑缺血诱导了Src蛋白激酶Ser和Tyr 4 16磷酸化及其与Pyk2结合 ,它们参与了Src的激活和NMDA受体功能的自调。     

Lyn modulates Claudin-2 expression and is a therapeutic target for breast cancer liver metastasis

Claudin-2 enhances breast cancer liver metastasis and promotes the development of colorectal cancers. The objective of our current study is to define the regulatory mechanisms controlling Claudin-2 expression in breast cancer cells.We evaluated the effect of several Src Family Kinase (SFK) inhibitors or knockdown of individual SFK members on Claudin-2 expression in breast cancer cells. We also assessed the potential effects of pan-SFK and SFK-selective inhibitors on the formation of breast cancer liver metastases. This study reveals that pan inhibition of SFK signaling pathways significantly elevated Claudin-2 expression levels in breast cancer cells. In addition, our data demonstrate that pan-SFK inhibitors can enhance breast cancer metastasis to the liver. Knockdown of individual SFK members reveals that loss of Yes or Fyn induces Claudin-2 expression; whereas, diminished Lyn levels impairs Claudin-2 expression in breast cancer cells. The Lyn-selective kinase inhibitor, Bafetinib (INNO-406), acts to reduce Claudin-2 expression and suppress breast cancer liver metastasis.Our findings may have major clinical implications and advise against the treatment of breast cancer patients with broad-acting SFK inhibitors and support the use of Lyn-specific inhibitors.     

Cholecystokinin activation of 70-kDa S6 kinase in exocrine pancreas

Cholecystokinin (CCK), a known mitogen for the exocrine pancreas, is shown to activate 70-kDa S6 kinase in isolated pancreatic acini. In this study, we examined the kinetics and cellular mechanisms of CCK-induced p70 S6 kinase activation in vivo and in vitro. Fasted mice were intraperitoneally injected with 0.01–10 g/kg CCK analoge cerulein. Cerulein caused a concentration-dependent activation of p70 S6 kinase, with the maximal effect at 1–10 g/kg. After 1 g/kg cerulein administration, the kinase activity was increased at 5 min, peaked at 10 min, and subsequently decreased. Cerulein also caused a rapid and transient activation of Src. Prior administration of the tyrosine kinase inhibitor herbimycin A compeletely inhibited cerulein-induced Src activation, while the inhibition of p70 S6 kinase activity was partial. Similar results were obtained with pancreatic acinar cell line AR42J cells. These results suggest that tyrosine kinases, including Src as a possible candidate, are partly implicated in the signaling pathway of CCK-induced p70 S6 kinase activation in the exocrine pancreas.     

Relationship between the in vitro response of dendritic cells to Lactobacillus and prevention of tumorigenesis in the mouse

BACKGROUND: Some strains of lactobacilli stimulate immune cells, yet little is known about their potency in cancer prevention. We have previously reported that Lactobacillus casei Shirota (LcS) suppresses murine tumorigenesis through immune modulation. In this study, differences were compared among six representative strains of lactobacilli in regard to their ability to stimulate bone marrow cell-derived dendritic cells (BMDCs) in vitro and tumor suppression in vivo. METHODS: BM-DCs were cocultured with a Lactobacillus strain in vitro, and the interleukin (IL)-12 released into the culture supernatant was measured by enzyme-linked immunosorbent assay. Tumors were chemically induced by a single subcutaneous injection of 3-methylcholanthrene (MC) in BALB/c mice. The test diets containing Lactobacillus were given from the day of the MC injection, and the tumor incidences were monitored. Peyer's patches were dissected from Lactobacillus-fed mice, and the status of c-Src, a regulator of DCs, in Peyer's patch cells was examined by Western blotting. RESULTS: In the coculture system, L. fermentum FERM P-13857 and LcS potently elicited IL-12 production. LcS but not the other strains of lactobacilli showed tumor suppression. The inactive form of c-Src, phosphorylated at Tyr527, was dominantly detected in Peyer's patches resected from L. fermentum FERM P-13857-fed mice compared with LcS-fed mice. CONCLUSIONS: The responses of DCs may be associated with tumor suppression by an ingested Lactobacillus strain.