Results: 5

1.
Fig. 3

Fig. 3. Regulation of TSC2 ubiquitination by Pam. From: Pam (Protein associated with Myc) functions as an E3 Ubiquitin ligase and regulates TSC/mTOR signaling.

(A) Ubiquitination of TSC2 by Pam is independent of its phosphorylation status. HEK293T cells were transfected with either wild type or mutant TSC2 (S939A/T1462A, S1798A, and S540/664A), along with Pam F3-Myc and Ub-HA as indicated. IPs performed with anti-FLAG or anti-Xpress antibody were analyzed with anti-HA antibody for ubiquitinated TSC2. (B) TSC1 protects TSC2 from ubiquitination by Pam. HEK293T cells were transfected with either FLAG-TSC2 (wild type) or disease-associated TSC mutants (R611Q and R905Q), along with Pam F3-Myc, TSC1-FLAG, and Ub-HA. TSC1 inhibits Pam F3 induced ubiquitination of wild type and R905Q mutant, but not R611Q mutant.

Sangyeul Han, et al. Cell Signal. ;20(6):1084-1091.
2.
Fig. 5

Fig. 5. Pam RNAi results in a reduction of P-S6 levels. From: Pam (Protein associated with Myc) functions as an E3 Ubiquitin ligase and regulates TSC/mTOR signaling.

(A) Co-transfection of E19 rat hippocampal cultures four days post-dissociation with both eGFP-N1 and an RNAi against PAM caused a reduction in somatic phosphorylated S6 ribosomal protein (P-S6) levels, compared to co-transfection of eGFP-N1/pSuper backbone at 4 DPT. Arrows indicate transfected neurons. Representative images. Scale Bar, 20µm. (C) Cumulative distribution of P-S6 immunostaining in eGFP-N1/pSuper-transfected or eGFP-N1/Pam RNAi-transfected hippocampal neurons at 4 DPT (two independent experiments, pSuper (n=40), Pam RNAi (n=40), p<0.01

Sangyeul Han, et al. Cell Signal. ;20(6):1084-1091.
3.
Fig. 4

Fig. 4. Pam regulates mTOR signaling through TSC2 downregulation in rat neurons. From: Pam (Protein associated with Myc) functions as an E3 Ubiquitin ligase and regulates TSC/mTOR signaling.

(A) Pam RNAi in rat cerebral cortical neurons resulted in an increase of TSC2 levels and subsequent downregulation of P-S6 and P-S6K levels. Dissociated cortical neuronal cells (5 DIV) from E18 rat were infected with lentiviruses expressing either control or Pam RNAi. At 9 DIV, cell lysates were prepared and analyzed by western analysis. (B) Quantitation of TSC2 levels in control and Pam RNAi groups (mean ± SEM; n=3, t-test, *p<0.05). Band intensities from western blots were analyzed using scanned images by densitometer (Bio-Rad). TSC2 levels were normalized against GAPDH levels. (C) Quantitation of P-S6 levels in control and Pam RNAi groups (mean ± SEM; n=3, t-test, *p<0.05). Band intensities were analyzed as described in B. P-S6 levels were normalized against total S6 levels.

Sangyeul Han, et al. Cell Signal. ;20(6):1084-1091.
4.
Fig. 2

Fig. 2. The RZF domain of Pam is essential for self-ubiquitination and TSC2 ubiquitination. From: Pam (Protein associated with Myc) functions as an E3 Ubiquitin ligase and regulates TSC/mTOR signaling.

(A) Consensus sequence of RZF domain and sequence alignment of RZF domains from human Pam, Mdm2 and Parkin. C=cysteine, H=histidine, X=any amino acid. (B) Mutation of three different cysteine residues (Cys-4394, Cys-4409, and Cys-4417) in the RZF domain of Pam (PamF3-3A) resulted in significant loss of Pam self-ubiquitination compared to Pam F3. IPs performed with anti-Myc antibodies were analyzed to detect ubiquitinated Pam F3 using anti-HA antibody. (C) Increased half-life of Pam F3-3A mutant. Twenty hours post-transfection with either Pam F3-Myc or Pam F3-3A-Myc, HEK293T cells were treated with cycloheximide for up to 24 hr to inhibit protein synthesis. (D) The effect of the RZF domain mutations on TSC2 ubiquitination. Pam F3-3A displays a reduction in TSC2 ubiquitination when compared with Pam F3. (E) TSC2 is downregulated by Pam F3.

Sangyeul Han, et al. Cell Signal. ;20(6):1084-1091.
5.
Fig. 1

Fig. 1. Pam interacts with ubiquitin-conjugating E2 enzymes and ubiquitinates TSC2. From: Pam (Protein associated with Myc) functions as an E3 Ubiquitin ligase and regulates TSC/mTOR signaling.

(A) Schematic diagram of Pam fragments. Pam F1 (aa 1–1669) contains two regulator of chromatin condensation (RCC)-homology domains (RHD1 and RHD2, residues 498–740 and 874–1065). Pam F2 (aa 1409–3568) contains a Myc-binding domain (aa 2413–2712) and Pam F3 (aa 3448–4641) possesses a RING finger domain (aa 4354–4440). (B) Carboxyl terminal Pam (Pam F3) binds to UbcH5a, UbcH5c and UbcH7 ubiquitin-conjugating E2 enzymes. Pull-down assays were carried out using recombinant E2 enzymes and HEK293T cell lysates transfected with Pam F3-Myc. (C) Pam F3 containing the RZF domain enhances ubiquitination of TSC2. HEK293T cells were transfected with TSC2-FLAG, Pam F3-Myc or Ub-HA. Immunoprecipitation (IP) performed with the anti-FLAG antibody was subjected to western analysis and ubiquitinated TSC2 was detected using the anti-HA antibody. (D) Tuberin, but not p53, is ubiquitinated by Pam. IPs were subjected to western analysis using anti-HA, anti-TSC2, or anti-p53 antibodies. Note the lack of ubiquitinated products in cells transfected with p53. However, ubiquitinated TSC2 was detected in cells transfected with Pam F3 and TSC2.

Sangyeul Han, et al. Cell Signal. ;20(6):1084-1091.

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