Journal: The Journal of Biological Chemistry

Article Title: A-kinase anchoring protein 8L interacts with mTORC1 and promotes cell growth

doi: 10.1074/jbc.AC120.012595

Figure Lengend Snippet: AKAP8L is an mTORC1-interacting protein. A, Raptor interacts with AKAP8L. Shown is co-immunoprecipitation of FLAG-tagged AKAP8L with HA-tagged Raptor. B, co-immunoprecipitation of HA-tagged Raptor with FLAG-tagged AKAP8L. C, co-immunoprecipitation of endogenous AKAP8L with endogenous Raptor. D, co-immunoprecipitation of HA-tagged Raptor with endogenous AKAP8L. E, schematic of the domains of AKAP8L and AKAP8. YG, YG-rich domain; FG, FG repeat region; NLS, nuclear localization sequence. F, mTORC1 interacts with AKAP8L. Shown is co-immunoprecipitation of HA-tagged Raptor and endogenous mTORC1 components with FLAG-tagged AKAP8L. IP, immunoprecipitation; EV, empty vector.

Article Snippet: For the generation of AKAP8L KO, HEK293A cells stably expressing FLAG-tagged full-length AKAP8L or amino acid region 247–646, a lentiviral vector (Addgene, #52962) encoding FLAG-AKAP8L full-length or FLAG-AKAP8L amino acid region 247–646 was transfected in HEK293A cells with packaging plasmids (Addgene, #12259 and #12260), and the produced virus was collected from the medium 48 h after transfection.

Techniques: Immunoprecipitation, Sequencing, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: A-kinase anchoring protein 8L interacts with mTORC1 and promotes cell growth

doi: 10.1074/jbc.AC120.012595

Figure Lengend Snippet: The N terminus of AKAP8L is required to interact with mTORC1. A, schematic of AKAP8L truncations generated based on relevant domains. B, AKAP8L amino acid region 247–646 does not interact with Raptor. Shown is co-immunoprecipitation of HA-tagged Raptor with the indicated FLAG-tagged AKAP8L truncations. C, AKAP8L amino acid region 63–247 interacts with Raptor. Shown is co-immunoprecipitation of FLAG-tagged AKAP8L amino acid region 63–247 with HA-tagged Raptor. IP, immunoprecipitation; FL, full-length; RFP, red fluorescent protein.

Article Snippet: For the generation of AKAP8L KO, HEK293A cells stably expressing FLAG-tagged full-length AKAP8L or amino acid region 247–646, a lentiviral vector (Addgene, #52962) encoding FLAG-AKAP8L full-length or FLAG-AKAP8L amino acid region 247–646 was transfected in HEK293A cells with packaging plasmids (Addgene, #12259 and #12260), and the produced virus was collected from the medium 48 h after transfection.

Techniques: Generated, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: A-kinase anchoring protein 8L interacts with mTORC1 and promotes cell growth

doi: 10.1074/jbc.AC120.012595

Figure Lengend Snippet: AKAP8L interacts with mTORC1 in the cytoplasm. A, AKAP8L localizes to the cytoplasm and nucleus. Expression of FLAG-tagged AKAP8L in mouse embryonic fibroblast cells. Red, FLAG-tagged AKAP8L (1.0 μg). B, expression of FLAG-tagged AKAP8L in the cytoplasmic and nuclear fractions of HEK293A cells. C, expression of endogenous AKAP8L in the cytoplasmic and nuclear fractions of HEK293A cells. D, Raptor interacts with AKAP8L in the cytoplasm. Co-immunoprecipitation of endogenous AKAP8L with HA-tagged Raptor. E, AKAP8L interacts with RIα. Co-immunoprecipitation of mCherry-tagged RIα with FLAG-tagged AKAP8L. F, Raptor interacts with AKAP8L and PKA machinery. Co-immunoprecipitation of FLAG-tagged AKAP8L, mCherry-tagged RIα, and FLAG-tagged PKA Catα with HA-tagged Raptor. IP, immunoprecipitation. s.e., short exposure; l.e., long exposure.

Article Snippet: For the generation of AKAP8L KO, HEK293A cells stably expressing FLAG-tagged full-length AKAP8L or amino acid region 247–646, a lentiviral vector (Addgene, #52962) encoding FLAG-AKAP8L full-length or FLAG-AKAP8L amino acid region 247–646 was transfected in HEK293A cells with packaging plasmids (Addgene, #12259 and #12260), and the produced virus was collected from the medium 48 h after transfection.

Techniques: Expressing, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: A-kinase anchoring protein 8L interacts with mTORC1 and promotes cell growth

doi: 10.1074/jbc.AC120.012595

Figure Lengend Snippet: AKAP8L regulates protein translation, cell size, and proliferation. A, depletion of AKAP8L decreases global protein synthesis. AKAP8L KO cells were incubated in methionine and cysteine-free Dulbecco's modified Eagle's medium for 1 h. Cycloheximide (CHX) treatment for 1 h was used as a positive control. 35S-labeled l-methionine and l-cysteine mix was then added to the medium for 10 min, and newly synthesized proteins were detected by autoradiography. Values are displayed as means ± S.D. (error bars). Significance was analyzed using Student's t test. B, loss of AKAP8L reduces the number of polysomes. AKAP8L KO cells with or without stably expressing full-length FLAG-tagged AKAP8L were subjected to polysome profiling. Negative control WT cells were treated with 100 nm Torin1 for 1 h. A representative image is shown from three biological replicates. C, loss of AKAP8L reduces cell size. The size of AKAP8L KO cells with or without stably expressing full-length FLAG-tagged AKAP8L or FLAG-tagged AKAP8L 247–646 was measured using a Coulter counter. Samples with the closest value to the mean are plotted as a representative image. Significance (p) was as follows: sgGFP versus sgAKAP8L#1 + FLAGEV, <0.01; sgGFP versus FLAGAKAP8L 247–646, <0.01; sgAKAP8L#1 + FLAGAKAP8L versus sgAKAP8L#1 + FLAGEV, <0.001; sgAKAP8L#1 + FLAGAKAP8L versus sgAKAP8L#1 + FLAGAKAP8L 247–646, <0.001. Significance was analyzed using Student's t test. The number of biological repeats is n ≥ 3. The number of technical repeats of each sample is n = 3 per experiment. D, loss of AKAP8L reduces cell proliferation. AKAP8L KO cells with or without stably expressing full-length FLAG-tagged AKAP8L or FLAG-tagged AKAP8L 247–646 were counted on the indicated days using trypan blue and a Bio-Rad automated cell counter. Values are displayed as means ± S.D. Significance was analyzed using Student's t test. The number of biological repeats is n ≥ 3. The number of technical repeats of each sample is n = 3 per experiment. E, working model of AKAP8L regulating translation, cell size, and proliferation. Significance is indicated as follows. **, p < 0.01; ***, p < 0.001.

Article Snippet: For the generation of AKAP8L KO, HEK293A cells stably expressing FLAG-tagged full-length AKAP8L or amino acid region 247–646, a lentiviral vector (Addgene, #52962) encoding FLAG-AKAP8L full-length or FLAG-AKAP8L amino acid region 247–646 was transfected in HEK293A cells with packaging plasmids (Addgene, #12259 and #12260), and the produced virus was collected from the medium 48 h after transfection.

Techniques: Incubation, Modification, Positive Control, Labeling, Synthesized, Autoradiography, Stable Transfection, Expressing, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: A-kinase anchoring protein 8L interacts with mTORC1 and promotes cell growth

doi: 10.1074/jbc.AC120.012595

Figure Lengend Snippet: The N terminus of AKAP8L is required to interact with mTORC1. A, schematic of AKAP8L truncations generated based on relevant domains. B, AKAP8L amino acid region 247–646 does not interact with Raptor. Shown is co-immunoprecipitation of HA-tagged Raptor with the indicated FLAG-tagged AKAP8L truncations. C, AKAP8L amino acid region 63–247 interacts with Raptor. Shown is co-immunoprecipitation of FLAG-tagged AKAP8L amino acid region 63–247 with HA-tagged Raptor. IP, immunoprecipitation; FL, full-length; RFP, red fluorescent protein.

Article Snippet: For the generation of AKAP8L KO, HEK293A cells stably expressing FLAG-tagged full-length AKAP8L or amino acid region 247–646, a lentiviral vector (Addgene, #52962) encoding FLAG-AKAP8L full-length or FLAG-AKAP8L amino acid region 247–646 was transfected in HEK293A cells with packaging plasmids (Addgene, #12259 and #12260), and the produced virus was collected from the medium 48 h after transfection.

Techniques: Generated, Immunoprecipitation