Display Settings:

Items per page

Results: 8

1.
FIG. 8

FIG. 8. From: Anisomycin Selectively Desensitizes Signalling Components Involved in Stress Kinase Activation and fos and jun Induction.

Location of the anisomycin-desensitized component relative to other intracellular signalling pathways. FRAP/TOR and p70/85S6k are included, as anisomycin clearly acts upstream of these enzymes. However, p70/85S6k desensitization could not be studied due to its strong and prolonged activation by anisomycin. The remaining circuitry involving MAP kinase subtypes and their upstream activators is discussed in detail in the Discussion. Note that anisomycin pretreatment desensitizes UV-stimulated JNK/SAPK activation to about 10% of the normal response, indicating that the anisomycin-desensitized component may be a major but not exclusive mediator of this response.

Catherine A. Hazzalin, et al. Mol Cell Biol. 1998 April;18(4):1844-1854.
2.
FIG. 1

FIG. 1. From: Anisomycin Selectively Desensitizes Signalling Components Involved in Stress Kinase Activation and fos and jun Induction.

Chemical structure of anisomycin (2-p-methoxyphenylmethyl-3-acetoxy-4-hydroxypyrrolidine). This bacterial compound (from Streptomyces griseolus and S. roseochromogenes [54]) was originally identified as an antibiotic against certain protozoa and fungi, which led to proposed clinical uses as a topical anticandidal and antiamoebic drug in humans. The pyrrolidine ring is important for the translational inhibitory activity of anisomycin; acetylation of the nitrogen or deacetylation at the 3′ position inhibits this activity (30). The signalling ability of anisomycin is also crucially dependent on the acetyl group; deacetylanisomycin compares very poorly with anisomycin in activating kinases and inducing fos and jun genes (26a).

Catherine A. Hazzalin, et al. Mol Cell Biol. 1998 April;18(4):1844-1854.
3.
FIG. 7

FIG. 7. From: Anisomycin Selectively Desensitizes Signalling Components Involved in Stress Kinase Activation and fos and jun Induction.

Anisomycin desensitization in the presence of transcriptional inhibitors. Normal C3H 10T½ cells (lanes 1 to 4) or cells in which transcription was blocked with actinomycin D (20 ng/ml; lanes 5 to 8) were pretreated with subinhibitory anisomycin (sAn; 50 ng/ml; lanes 3, 4, 7, and 8) for 3 h or not pretreated (−; lanes 1, 2, 5, and 6) as indicated in the top line (pretreat) prior to restimulation with subinhibitory anisomycin (sAn; 50 ng/ml; lanes 2, 4, 6, and 8) for 1 h, as shown in the second line (2nd stim), C, control (unstimulated; lanes 1 and 5); − (lanes 3 and 7), no restimulation. The upper panels show solid-phase kinase assays of GST-cJun1-79 binding kinases; the lower panels show in-gel kinase assays of MAPKAP K-2 phosphorylation exactly as described for Fig. 5 and 6.

Catherine A. Hazzalin, et al. Mol Cell Biol. 1998 April;18(4):1844-1854.
4.
FIG. 5

FIG. 5. From: Anisomycin Selectively Desensitizes Signalling Components Involved in Stress Kinase Activation and fos and jun Induction.

Effects of anisomycin desensitization on anisomycin-stimulated activation of JNK/SAPKs and p38/RK. C3H 10T½ cells were pretreated (pretreat) with subinhibitory anisomycin (sAn; 50 ng/ml; lanes 4 to 6) for 3 h or not pretreated (−; lanes 1 to 3) prior to restimulation (2nd stim) with anisomycin at either an inhibitory (An; 10 μg/ml; lanes 2 and 5) or subinhibitory (sAn; 50 ng/ml; lanes 3 and 6) concentration for 1 h or no restimulation (−; lane 4). C, control (unstimulated; lane 1). (A) Solid-phase kinase assay of GST-cJun1-79 binding kinases. Whole-cell extracts were prepared from cells stimulated as described above, and GST-cJun1-79 binding kinases were isolated and assayed by in vitro phosphorylation of the fusion protein as described in Materials and Methods. The position of GST-cJun1-79 is indicated. (B) In-gel kinase assay of MAPKAP K-2 phosphorylation. Total lysates from cells stimulated as described above and prepared as described in Materials and Methods were analyzed by SDS-polyacrylamide gel electrophoresis and in-gel kinase assay in gels containing 200 μg of copolymerized poly-Glu/Tyr per ml. The positions of the 45- and 55-kDa forms of MAPKAP K-2 are indicated (10).

Catherine A. Hazzalin, et al. Mol Cell Biol. 1998 April;18(4):1844-1854.
5.
FIG. 6

FIG. 6. From: Anisomycin Selectively Desensitizes Signalling Components Involved in Stress Kinase Activation and fos and jun Induction.

Effects of anisomycin desensitization on JNK/SAPK and p38/RK activation by diverse stimuli. C3H 10T½ cells were not pretreated (A and B) or pretreated (C and D) with subinhibitory anisomycin (sAn; 50 ng/ml) for 3 h as indicated in the top line (pretreat) prior to restimulation with either an inhibitory (An; 10 μg/ml; lanes 2, 10, 14, and 22) or subinhibitory (sAn; 50 ng/ml; lanes 3 and 15) concentration of anisomycin for 1 h, EGF (E; 50 ng/ml, 15 min; lanes 4 and 16), bFGF (F; 20 ng/ml, 15 min; lanes 5 and 17), TNF-α (Tα; 10 ng/ml, 15 min; lanes 6 and 18), TPA (T; 100 nM, 15 min; lanes 7 and 19), UV radiation (UV; 200 J/m2, 30 min; lanes 8 and 20), sorbitol (Srb; 0.5 M, 30 min; lanes 11 and 23), or sodium chloride (NaCl; 0.5 M, 30 min; lanes 12 and 24) as shown in the second line (2nd stim). Lanes 1, 9, 13, and 21, unstimulated cells; − (lane 9), no restimulation after pretreatment. (A and C) Solid-phase kinase assays of GST-cJun1-79 binding kinases. Sequestration and kinase assays for GST-cJun1-79 were done exactly as described in the legend to Fig. 5. (B and D) In-gel kinase assay of MAPKAP K-2 activity. Total lysates from cells stimulated as described above were analyzed by in-gel kinase assay in gels containing 200 μg of copolymerized poly-Glu/Tyr per ml. The positions of the 45- and 55-kDa forms of MAPKAP K-2 are indicated.

Catherine A. Hazzalin, et al. Mol Cell Biol. 1998 April;18(4):1844-1854.
6.
FIG. 4

FIG. 4. From: Anisomycin Selectively Desensitizes Signalling Components Involved in Stress Kinase Activation and fos and jun Induction.

Effects of UV irradiation on fos and jun gene induction responses to specific stimuli. (A) Northern blot analysis of c-fos, fosB, c-jun, junB, and junD induction following pretreatment with UV radiation. Nontreated (−; lanes 1, 2, 8 to 12, 14, 16, and 18) or UV-irradiated (UV; 200 J/m2; lanes 3 to 7, 13, 15, 17, and 19) C3H 10T½ cells were incubated for 3 h, followed by restimulation (2nd stim) with UV radiation (UV; 200 J/m2, 45 min; lanes 2, 4, 12, and 13), subinhibitory anisomycin (sAn; 25 ng/ml, 45 min; lanes 5 and 8), anisomycin (An; 10 μg/ml, 45 min; lane 6), TNF-α (Tα; 5 ng/ml, 30 min; lanes 7 and 9), EGF (E; 50 ng/ml, 30 min; lanes 10, 14, and 15), bFGF (F; 20 ng/ml, 30 min; lanes 16 and 17); TPA (T; 100 nM, 30 min; lanes 18 and 19), or no secondary stimulus (−; lane 3). C, control (unstimulated; lanes 1 and 11). GAPDH was used as a loading control, and RNA prepared from EGF-stimulated cells (E; 50 ng/ml; 30 min; lanes 10 and 14) was included as a control to standardize between experiments and blots. (B) Inhibition of anisomycin-, EGF-, and EGF-anisomycin-stimulated c-fos, fosB, c-jun, junB, and junD induction by cotreatment with UV radiation. Northern blot analysis was performed for total RNA from quiescent confluent C3H 10T½ cells treated with UV radiation (+; 200 J/m2; lanes 2, 4, 6, and 8) or not treated (−; lanes 1, 3, 5, and 7) and then immediately stimulated with anisomycin (An; 10 μg/ml, 45 min; lanes 3 and 4), EGF plus anisomycin (EAn; 50 ng/ml and 10 μg/ml, respectively, 45 min; lanes 5 and 6), or EGF (50 ng/ml, 30 min; lanes 7 and 8). C, control (unstimulated; lane 1). GAPDH was used as a loading control.

Catherine A. Hazzalin, et al. Mol Cell Biol. 1998 April;18(4):1844-1854.
7.
FIG. 3

FIG. 3. From: Anisomycin Selectively Desensitizes Signalling Components Involved in Stress Kinase Activation and fos and jun Induction.

Desensitization of fos and jun induction by subinhibitory anisomycin and TNF-α. (A) Northern blot analysis of c-fos, fosB, c-jun, junB, and junD gene expression. C3H 10T½ cells were pretreated for 3 h (agents indicated in top line, marked “pretreat”) with subinhibitory anisomycin (sAn; 25 ng/ml; lanes 3 to 6 and 22 to 24) or TNF-α (TNF-α; 10 ng/ml; lanes 8 to 11; Tα5, 5 ng/ml; lane 16) or not pretreated (−; lanes 1, 2, 7, 12 to 15, and 17 to 21), followed by restimulation (2nd stim) with either EGF (E; 50 ng/ml, 30 min; lanes 4, 9, 12, 17, and 19), subinhibitory anisomycin (sAn; 25 ng/ml, 45 min; lanes 2, 5, 10, 21, and 23), inhibitory anisomycin (An; 10 μg/ml, 45 min; lane 24), TNF-α (Tα; 10 ng/ml; lanes 6, 7, and 11; Tα5; 5 ng/ml, 30 min; lane 15), UV radiation (UV; 200 J/m2, 45 min; lanes 14 and 16), bFGF (F; 20 ng/ml, 30 min, lanes 20 and 22), or no secondary stimulus (−; lanes 3 and 8). C, control (unstimulated; lanes 1, 13, and 18). GAPDH was used as a loading control, and RNA prepared from EGF-stimulated cells (E; 50 ng/ml, 30 min; lanes 12, 17, and 19) was included as a control to standardize between experiments and blots. (B) Quantification of the extent of desensitization of c-fos, fosB, c-jun, and junB gene induction responses following subinhibitory anisomycin and TNF-α pretreatment. Autoradiographs of the Northern blots shown in Fig. 3A or not shown (see below) were subjected to densitometric analysis, and the absorbance values were corrected for variations in loading by using GAPDH. Agents used for pretreatment (pretreat) are indicated at the top, while agents used to restimulate cells are indicated along the x axis (2nd stim). All desensitization data are expressed as a percentage of the normal response of that particular gene in nondesensitized cells, the normal response (100%) being indicated by a dashed line and the cutoff being 200%. The extent of any restimulation response greater than 200% is indicated by the exact number above the relevant column. Northern blot analyses of some data are not shown in panel A: subinhibitory anisomycin pretreatment (25 ng/ml; 3 h) and restimulation with TPA (100 nM; 30 min) or UV radiation (200 J/m2; 45 min).

Catherine A. Hazzalin, et al. Mol Cell Biol. 1998 April;18(4):1844-1854.
8.
FIG. 2

FIG. 2. From: Anisomycin Selectively Desensitizes Signalling Components Involved in Stress Kinase Activation and fos and jun Induction.

Desensitization of fos and jun induction by EGF and bFGF. (A) Northern blot analysis of c-fos, fosB, c-jun, junB, and junD gene expression. C3H 10T½ cells were pretreated for 3 h (agents indicated in top line, marked “pretreat”) with EGF (50 ng/ml; lanes 3 to 5, 10, 13, and 14) or bFGF (20 ng/ml; lanes 16 to 19) or not pretreated (top line, marked “-”; lanes 1, 2, 6 to 9, 11, 12, 15, and 20), followed by restimulation (marked “2nd stim”) with either EGF (E; 50 ng/ml, 30 min; lanes 2, 3, 9, 12, 13, and 16), bFGF (F; 20 ng/ml; 30 min; lanes 14, 15, and 17), subinhibitory anisomycin (sAn; 25 ng/ml, 45 min; lanes 4, 6, 18, and 20), inhibitory anisomycin (An; 10 μg/ml, 45 min; lanes 5 and 19), or UV radiation (UV; 200 J/m2, 45 min; lanes 8 and 10). C, control (unstimulated; lanes 1, 7, and 11). GAPDH was used as a loading control, and RNA prepared from EGF-stimulated cells (E; 50 ng/ml, 30 min; lanes 2, 9, and 12) was included as a control to standardize between experiments and blots. Northern blot analyses of some data are not shown: EGF pretreatment (50 ng/ml; 3 h) and restimulation with TPA (100 nM; 30 min) or UV radiation (200 J/m2; 45 min). (B) Quantification of the extent of desensitization of c-fos, fosB, c-jun, and junB gene induction responses following EGF and bFGF pretreatment. Autoradiographs of the Northern blots shown in Fig. 2A or not shown (see below) were subjected to densitometric analysis, and the absorbance values were corrected for variations in loading, using GAPDH. Agents used for pretreatment (pretreat) are indicated at the top, while agents used to restimulate cells are indicated along the x axis (2nd stim). All desensitization data are expressed as a percentage of the normal response of that particular gene in nondesensitized cells, the normal response (100%) being indicated by a dashed line and the cutoff being 200%. The extent of any restimulation response greater than 200% (in this case TPA) is indicated by the exact number above the relevant column. Asterisks indicate points where the normal response is zero, but transcripts are detectable in the desensitized cells; the extent cannot therefore be quantified.

Catherine A. Hazzalin, et al. Mol Cell Biol. 1998 April;18(4):1844-1854.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk