Interleukin-32δ interacts with IL-32β and inhibits IL-32β-mediated IL-10 production

There is growing evidence for multifunctional properties of IL-32. We previously demonstrated that IL-32β upregulates IL-10 production through the association with PKCδ. In this study, we examined the effects of other IL-32 isoforms on IL-10 production. We found that IL-32δ decreased IL-10 production and investigated the inhibitory mechanism of IL-32δ. We showed that IL-32δ suppressed IL-32β binding to PKCδ by interacting with IL-32β. The inhibitory effect of IL-32δ on IL-32β association with PKCδ was further verified by immuno-fluorescence staining. The co-localization of IL-32β and PKCδ around the nuclear membrane was disrupted by IL-32δ. Our data therefore indicate that IL-32δ plays an inhibitory role against IL-32β function, which also suggests that IL-32 may be regulated by its own isoform.     

32P-labeling of bovine tryptophanyl-tRNA synthetase with [32P]pyrophosphate

The covalent derivative of the tryptophanyl-tRNA synthetase obtained under the action of³²PP_i contains one mole of the covalently bound pyrophosphate (or 2 moles of orthophosphate) per mole of dimeric enzyme. Dephosphorylation with alkaline phosphatase causes practically no changes of enzymatic activity although the enzyme looses its ability to bind PP_i.Enzymes tryptophanyl-tRNA synthetase (EC 6.1.1.2), alkaline phosphatase (EC 3.1.3.1), inorganic pyrophosphatase (EC 3.6.1.1)     

Induction of tight junctions in human connexin 32 (hCx32)-transfected mouse hepatocytes: connexin 32 interacts with occludin

Small gap junction plaques are associated with tight junction strands in some cell types including hepatocytes and it is thought that they may be closely related to tight junctions and the establishment of cell polarity. In order to examine roles of gap junctions in regulating expression and structure of tight junctions, we transfected human Cx32 cDNA into immortalized mouse hepatocytes (CHST8 cells) which lack endogenous Cx32 and Cx26. Immunocytochemistry revealed that endogenous integral tight junction protein occludin was strongly localized and was colocalized with Cx32 at cell borders in transfectants, whereas neither was detected in parental cells. In Northern blots, mRNAs encoding occludin and the other integral tight junction proteins, claudin-1 and -2, were induced in the transfectants compared to parental cells. In Western blots, occludin protein was increased in the transfectants compared to parental cells, and binding of occludin to Cx32 protein was demonstrated by immunoprecipitation. In freeze fracture of the transfectants, tight junction strands were more numerous and complex compared to parental cells, and small gap junction plaques appeared within induced tight junction strands. Nevertheless, no change in barrier function of tight junctions was observed. These results indicate that in hepatocytes, gap junction, and tight junction expression are closely coordinated, and that Cx32 may play a role in regulating occludin expression.     

Phosphorylated retinoblastoma protein complexes with pp32 and inhibits pp32-mediated apoptosis

The retinoblastoma gene product (Rb) is a tumor suppressor that affects apoptosis paradoxically. Most sporadic cancers inactivate Rb by preferentially targeting the pathway that regulates Rb phosphorylation, resulting in resistance to apoptosis; this contrasts with Rb inactivation by mutation, which is associated with high rates of apoptosis. How phosphorylated Rb protects cells from apoptosis is not well understood, but there is evidence that Rb may sequester a pro-apoptotic nuclear factor. pp32 (ANP32A) is a pro-apoptotic nuclear phosphoprotein, the expression of which is commonly increased in cancer. We report that hyperphosphorylated Rb interacts with pp32 but not with the closely related proteins pp32r1 and pp32r2. We further demonstrate that pp32-Rb interaction inhibits the apoptotic activity of pp32 and stimulates proliferation. These results suggest a mechanism whereby cancer cells gain both a proliferative and survival advantage when Rb is inactivated by hyperphosphorylation.