Focal Adhesion Kinase Directly Interacts with TSC2 Through Its FAT Domain and Regulates Cell Proliferation in Cashmere Goat Fetal Fibroblasts

Abstract

Focal adhesion kinase (FAK) is a cytoplasmic nonreceptor tyrosine kinase that senses a variety of extracellular signals, such as growth factors and integrins, to control the process of cell proliferation and metabolism. We cloned three goat FAK transcript variants (KM655805, KM658268, and KM658269) that encode 1052, 1006, and 962 amino-acid residue proteins. Bioinformatics analysis indicated that the putative FAK protein contains an FERM domain, a PTK domain, two Proline-rich regions, and a focal adhesion-targeting (FAT) domain. All the three transcript variants of FAK were detected in seven different goat tissues, and variant 1 had the most accumulation whereas variant 2 and variant 3 had lower accumulation. Treatment of goat fetal fibroblasts (GFbs) with a specific FAK inhibitor, TAE226, inhibited cell proliferation (p < 0.05) and induced damage to the cell morphology in a dose- and time-dependent manner. Further research demonstrated that FAK directly interacted with TSC2 (Tuberous sclerosis 2) tuberin domain through its C-terminus, which contains the complete FAT domain. In conclusion, our results indicated that FAK may be widely expressed in Cashmere goat tissues and its products participate in the mammalian target of rapamycin signaling pathway and cell proliferation through a direct interaction with TSC2 in GFBs.

A Novel Heterogalactan from Antrodia camphorata and Anti-Angiogenic Activity of Its Sulfated Derivative

“…Moreover, the remaining resonances at 69.50, 70.75, and 70.07 ppm were attributed to be C2, C3/C4, and C5 of 1,6-linked galactose, respectively. These data were in good accordance with previously reported results [15,18,33].…”

Phosphoproteomics Analysis Reveals a Pivotal Mechanism Related to Amino Acid Signals in Goat Fetal Fibroblast

“…The procedures used to isolate and characterize of GFbs were performed as previously described ( 33 ). GFb cell cultures were maintained as previously described ( 34 ). Briefly, GFb cells were cultured as monolayer cultures in Dulbecco's modified Eagle medium (DMEM)/F12 (GE Healthcare Life Sciences, Logan, Utah, U.S.A.) supplemented with 10% fetal bovine serum (Sigma-Aldrich, Saint Louis, Missouri, U.S.A.) and 1% penicillin-streptomycin (100×) (TransGen Biotech, Beijing, China).…”

The mTORC1/4EBP1/PPARγ Axis Mediates Insulin-Induced Lipogenesis by Regulating Lipogenic Gene Expression in Bovine Mammary Epithelial Cells

“…Then, equivalent amounts of protein lysate were incubated with anti-4EBP1 (1:50) or anti-His (1:50) (negative control) and inactive resin (negative control) in Millipore catch-and-release spin columns. Equivalent amounts of protein lysate were also evaluated by Western blot as a positive control for 4EBP1 and PPARγ, per reported methods. , …”

“…Diploid cells were then screened on an SD/-Leu/-Trp/X-a-Gal/AbA (DDO/X/A) plate, and the resulting blue colonies were transferred to an SD/-Leu/-Trp/-His/X-α-Gal/AbA (TDO/X/A) plate and then to an SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA (QDO/X/A) plate, followed by incubation for 3–6 days at 30 °C. The yeast two-hybrid screen was performed as described. , …”

“…Equivalent amounts of protein lysate were also evaluated by Western blot as a positive control for 4EBP1 and PPARγ, per reported methods. 35,36 A yeast two-hybrid screen was performed using the Yeastmaker Yeast Transformation System 2 and Matchmaker Gold Yeast Two-Hybrid System (Clontech Laboratories, Inc., Mountain View, CA) per the manufacturer's instructions. Reagents for the synthetically defined plate and prototroph and colorimetric screening were obtained from Clontech.…”