Guoxuan Li's research while affiliated with University of North Carolina at Chapel Hill and other places

What is this page?

This page lists the scientific contributions of an author, who either does not have a ResearchGate profile, or has not yet added these contributions to their profile.

It was automatically created by ResearchGate to create a record of this author's body of work. We create such pages to advance our goal of creating and maintaining the most comprehensive scientific repository possible. In doing so, we process publicly available (personal) data relating to the author as a member of the scientific community.

If you're a ResearchGate member, you can follow this page to keep up with this author's work.

If you are this author, and you don't want us to display this page anymore, please let us know.

Publications (2)

FIG. 1. PGE 1 and PGE 2 inhibit CIITA-mediated transactivation. P388D1 cells were transfected with the plasmid DNAs shown and treated with the indicated concentrations of either PGE 1 (A, C, and D) or PGE 2 (B, C, and E). Luciferase or CAT activity was determined as described in Materials and Methods. Transcriptional activity is expressed as a percentage relative to activity using untreated, wild-type Gal4CIITA (A, B, and C) or CIITA (D and E). Results are presented as the means standard deviations of three separate experiments.
FIG. 2. cAMP inhibits CIITA-mediated transactivation. Transcriptional activation by Gal4CIITA (A) or CIITA (B) was determined using P388D1 cells transfected with the indicated plasmid DNAs and cultured in the absence or presence of increasing concentrations of 8-bromo-cAMP as indicated. Relative activation (as a percentage) is shown as described for Fig. 1. Values are means standard deviations from three separate experiments.
FIG. 3. PKA ␣ inhibits CIITA-mediated transactivation. (A) PKA ␣ inhibits CIITA-mediated promoter activation. Gal4DRCAT and Gal4CIITA (bars 1 to 4) or ddDrCAT and p3FgCIITA (bars 5 and 6) together with plasmids encoding either the PKA ␣ or PKA ␤ catalytic subunit (c) were cotransfected into P388D1 cells. (B) PKA ␣ represses IFN- ␥ -induced promoter activity. P388D1 cells were transfected with 300DRLuc with or without cotransfection of PKA ␣ and cultured in the presence or absence of recombinant mouse IFN- ␥ (rIFN- ␥ ). (C) PKA ␣ inhibits CIITA-mediated transcription of endogenous class II MHC. P388D1 cells were transfected with empty vector or wild-type CIITA and treated as indicated. Cells were treated with IFN- ␥ (0.8 ng/ml), PGE 1 (1.0 ␮ M), or cAMP (1.0 ␮ M) or cotransfected with PKA ␣ as indicated. Expression of mRNA was determined by reverse transcription-PCR (see Materials and Methods). (D) PKA ␣ inhibits CIITA-mediated transactivation in monocytic cell lines. The monocytic cell lines U937 and RAW264 were cotransfected with 300DRLuc and CIITA with or without PKA ␣ using SuperFect (Qiagen). CAT (A) and luciferase (B and D) activities were determined at 36 h posttransfection. The data are the means Ϯ standard deviations of three experiments and are shown normalized to the activity of Gal4CIITA or CIITA on the Gal4DRCAT or 300DRLuc reporter, respectively (100%). 
FIG. 4. CIITA phosphorylation assay in vitro and in vivo with PKA. (a) Lane 1, CIITA immunopurified from transfected-cell lysate; lanes 2 to 4, in vitro-translated and immunopurified CIITA was tested for phosphorylation by PKA with negative controls; lanes 5 through 8, immunopurified CIITA from translation in vitro was assayed for phos- phorylation capability with kinases of cGMP-dependent protein ki- nase, protein kinase C, calmodulin-dependent protein kinase II, and growth-associated histone H1 kinase; lane 9, immunopurified CIITA from transiently transfected cells labeled with [ 32 P]orthophosphate in vivo; lane 10, immunopurified CIITA labeled with [ 33 P]orthophos- phate in vivo from P388D1 cells transiently cotransfected with CIITA and PKA ␣ . (b) Two-dimensional phosphoamino acid analysis using [ ␥ - 32 P]ATP-labeled CIITA in vitro obtained from lane 2. (c) Two- dimensional phosphoamino acid analysis using [ 33 P]orthophosphate- labeled CIITA in vivo obtained from lane 10. 
FIG. 5. Mapping of PKA phosphorylation sites in CIITA. (A) Di- agram of CIITA showing potential PKA phosphorylation sites, relative positions of CNBr cleavage sites, and the predicted molecular masses of each cleavage product. Potential PKA phosphorylated serine resi- dues are in boldface. (B) One-dimensional phosphopeptide mapping. Digestion of phosphorylated CIITA was performed with CNBr to generate peptides. Arrows, expected CNBr cleavage peptide frag- ments; the corresponding predicted molecular masses are shown. Pep- tides were separated by SDS-PAGE (16%). The observed molecular weights of 32 P-labeled fragments are based on migration of molecular weight standards. 

+2

Downregulation of CIITA Function by Protein Kinase A (PKA)-Mediated Phosphorylation: Mechanism of Prostaglandin E, Cyclic AMP, and PKA Inhibition of Class II Major Histocompatibility Complex Expression in Monocytic Lines
  • Article
  • Full-text available

August 2001

·

91 Reads

·

61 Citations

Molecular and Cellular Biology

Guoxuan Li

·

·

Xinsheng Zhu

·

Jenny P.-Y. Ting

Prostaglandins, pleiotropic immune modulators that induce protein kinase A (PKA), inhibit gamma interferon induction of class II major histocompatibility complex (MHC) genes. We show that phosphorylation of CIITA by PKA accounts for this inhibition. Treatment with prostaglandin E or 8-bromo-cyclic AMP or transfection with PKA inhibits the activity of CIITA in both mouse and human monocytic cell lines. This inhibition is independent of other transcription factors for the class II MHC promoter. These same treatments also greatly reduced the induction of class II MHC mRNA by CIITA. PKA phosphorylation sites were identified using site-directed mutagenesis and phosphoamino acid analysis. Phosphorylation at CIITA serines 834 and 1050 accounts for the inhibitory effects of PKA on CIITA-driven class II MHC transcription. This is the first demonstration that the posttranslational modification of CIITA mediates inhibition of class II MHC transcription.

Download
Share
FIG. 1. Transactivation of the DRA promoter by CIITA requires stereospecifically aligned and/or properly spaced W-X-Y elements. The transactivation of different DRA promoter mutants by CIITA compared with that by IFN-is shown. Fold activation is calculated as CAT expression in the presence of CIITA or IFN-divided by CAT expression in its absence. Mutated W, X, Y, or octamer sequence is indicated by an "X." W(X5)Y and W(X10)Y have insertions of 5 and 10 bp, respectively, between X and Y, while (W5)XY and (W10)XY have insertions of 5 and 10 bp, respectively, between W and X.
FIG. 2. CIITA interacts with the B and C subunits of NF-Y/CBF and with RFX5 in a cell-free system. (A) CIITA interacts with NF-YC and NF-YB. 35 S-labeled myc-tagged CIITA (mycCIITA) and 35 S-labeled FlagNF-YA (lanes 1 and 2), FlagNF-YB (lanes 4 and 5), or FlagNF-YC (lanes 7 and 8) were produced by cotranslation of these products. The minus signs in the chart indicate the presence of either an appropriate empty control plasmid or an appropriate Ig control. The mixtures were immunoprecipitated with anti-myc (9E10) antibody (lanes 1, 4, and 7) or preimmune serum (lanes 2, 5, and 8) and anti-mouse IgG agarose. In vitro-translated 35 S-labeled FlagNF-YA (lane 3), FlagNF-YB (lane 6), and FlagNF-YC (lane 9) were included to show the electrophoresis pattern of the expected translation products. The translated CIITA appears in the upper part of lanes 1, 4, and 7. Coprecipitated NF-YB (lane 4) and NF-YC (lane 7) are seen comigrating with the input product shown in lanes 6 and 9. (B) CIITA interacts with RFX5 in vitro. The experiment was performed similarly to that described for panel A, except that the in vitro-translated FlagCIITA was not radiolabeled, and only the RFX5 protein was radiolabeled. Lane 2 shows a weak band indicative of RFX5 which coprecipitated with the CIITA protein. Lane 3 shows the electrophoresis pattern of RFX5. 
FIG. 3. CIITA interacts with NF-YB–NF-YC and RFX5 in cells. (A and B) Cells (9 ϫ 10 5 ) were cotransfected with 3 ␮ g of mycCIITA or its corresponding control vector pcDNA3myc and 3 ␮ g of FlagNF-YA, FlagNF-YB, or FlagNF-YC as indicated. Cells were lysed with 1.5 ml of RIPA buffer 36 h after transfection, and 25 ␮ l of the samples was subjected to SDS–10% polyacrylamide gel elec- trophoresis resolution. The expression of mycCIITA and FlagNF-YA, FlagNF- YB, or FlagNF-YC was analyzed by Western blotting using either anti-Flag (M5) (A) or anti-myc (9E10) (B) antibodies, which respectively detect the NF-Y subunits or CIITA. IB, immunoblotting. (C) Half of the cell lysate described for panel A was incubated with 3.7 ␮ g of anti-myc (9E10) antibody for 1.5 h. The immunocomplexes were incubated at 4°C overnight with 15 ␮ l of anti-mouse IgG Dynabeads. The association of CIITA with NF-YA (lane 2), NF-YB (lane 4), or NF-YC (lane 6) was studied by Western blotting using the anti-Flag (M5) antibody. IP, immunoprecipitation. (D) Prolonged exposure of the blot in panel C shows a weak interaction between CIITA and NF-YA (asterisk). H and L designate heavy and light chains of Ig, respectively. (E) CIITA interacts with RFX5. Similar to the experiments performed for panels A to D, FlagRFX5 was cotransfected with either mycCIITA or pcDNA3myc. Cell lysate was immuno- precipitated using the anti-myc (9E10) antibody. The associated FlagRFX5 was detected by anti-Flag (M5) antibody immunoblotting. 
FIG. 4. CIITA residues 518 to 612 interact with NF-YB in cells. To map the domain of CIITA that is required for interaction with NF-YB, a series of FlagCIITA C- and N-terminal deletion mutants were constructed. Each sample was cotransfected with 3 ␮ g of FlagCIITA or its mutants and 3 ␮ g of a myc-tagged NF-YB subunit. The subsequent immunoprecipitation, electrophoresis, and Western blotting procedures were performed as described in the Fig. 3 legend. (A) Expression of NF-YB in total cell lysate was detected by immunoblotting with the rabbit anti-myc antibody. (B) Expression of FlagCIITA and its mutants in total lysate was confirmed by immunoblotting with the anti-Flag (M5) antibody. (C) FlagCIITA and its C-terminal deletion mutants were used to coprecipitate mycNF-YB, which was detected by rabbit anti-myc antibody. (D to F) The same as panels A to C, respectively, except that N-terminal deletion mutants were used. (G) The two constructs which delineated the residues within CIITA that interacted with NF-YB. The black area marks the overlapping residues shared by these two constructs. 
FIG. 5. CIITA residues 218 to 335 interact with NF-YC in cells. To map the domain of CIITA that is required for interaction with NF-YC, a series of FlagCIITA C- and N-terminal deletion mutants were constructed. The experiment was performed similarly to that for Fig. 4. (A) Expression of NF-YC in total cell lysate was detected by immunoblotting with the rabbit anti-myc antibody. (B) Expression of FlagCIITA and its mutants in total lysate was confirmed by immunoblotting with the anti-Flag (M5) antibody. (C) FlagCIITA and its C-terminal mutants were used to coprecipitate mycNF-YC, which was detected by rabbit anti-myc antibody. (D to F) The same as panels A to C, respectively, except that N-terminal mutants were used. (G) The two constructs which delineated the residues within CIITA which interacted with NF-YC. See the Fig. 3 legend for more details. 

+4

Transcriptional Scaffold: CIITA interacts with NF-Y, RFX, and CREB to cause stereospecific regulation of the Class II Major histocompatibility complex promoter

September 2000

·

105 Reads

·

194 Citations

Molecular and Cellular Biology

Xin-Sheng Zhu

·

·

Guoxuan Li

·

[...]

·

Scaffold molecules interact with multiple effectors to elicit specific signal transduction pathways. CIITA, a non-DNA-binding regulator of class II major histocompatibility complex (MHC) gene transcription, may serve as a transcriptional scaffold. Regulation of the class II MHC promoter by CIITA requires strict spatial-helical arrangements of the X and Y promoter elements. The X element binds RFX (RFX5/RFXANK-RFXB/RFXAP) and CREB, while Y binds NF-Y/CBF (NF-YA, NF-YB, and NF-YC). CIITA interacts with all three. In vivo analysis using both N-terminal and C-terminal deletion constructs identified critical domains of CIITA that are required for interaction with NF-YB, NF-YC, RFX5, RFXANK/RFXB, and CREB. We propose that binding of NF-Y/CBF, RFX, and CREB by CIITA results in a macromolecular complex which allows transcription factors to interact with the class II MHC promoter in a spatially and helically constrained fashion.

Download
Share

Citations (2)

... The deficiency of STAT1 in mice made them highly susceptible to bacterial and viral infections, ultimately leading to death [30][31][32]. The NFYC protein binds to the promoter of major histocompatibility complex (MHC) genes and modulates their transcription [33,34], and therefore plays important roles in immune responses against viruses and pathogens. Besides these two genes, several candidate genes related to high-altitude adaptation were identified (e.g., NABP1, which may be involved in the adaptation to radiation from high altitudes). ...

Reference:

Identification of Selection Signatures and Candidate Genes Related to Environmental Adaptation and Economic Traits in Tibetan Pigs
Transcriptional Scaffold: CIITA interacts with NF-Y, RFX, and CREB to cause stereospecific regulation of the Class II Major histocompatibility complex promoter

Molecular and Cellular Biology

Xin-Sheng Zhu

·

·

Guoxuan Li

·

[...]

·

... The activity of CIITA is regulated by various posttranslational modifications such as ubiquitination, phosphorylation, and acetylation. Phosphorylation is important for transcriptional activity, nuclear localization, multimerization, and protein-protein interactions, while acetylation is important for nuclear importation of CIITA (13)(14)(15)(16). Monoubiquitination of CIITA increases transcriptional activity, whereas polyubiquitination plays a role in nuclear translocation and degradation of CIITA (17,18). ...

Reference:

FBXO11 constitutes a major negative regulator of MHC class II through ubiquitin-dependent proteasomal degradation of CIITA
Downregulation of CIITA Function by Protein Kinase A (PKA)-Mediated Phosphorylation: Mechanism of Prostaglandin E, Cyclic AMP, and PKA Inhibition of Class II Major Histocompatibility Complex Expression in Monocytic Lines

Molecular and Cellular Biology

Guoxuan Li

·

·

Xinsheng Zhu

·

Jenny P.-Y. Ting