Биологические науки/5.Молекулярная  биология

Bulgakova O.V., Bersimbay R.I.

mTOR Complex 2 Is Required for the Development of Epithelial cells in Mice.

L.N. Gumilyov Eurasian National University, Kazakhstan

mTOR belongs to the family of phosphoinositide-3-kinase (PI3K)-related kinases (PIKKs), along with ATM, ATR, DNA-PK, and hSMG1[1]. All of these proteins have C-terminal protein kinase domains with similarity to the lipid kinase PI3K, thus giving the family its name mTOR forms two different protein complexes defined by the proteins to which it is bound, exerting different but related functions [2]. The mTOR complex 1 (mTORC1) is defined by the presence of Raptor, mLST8/GβL, Deptor and Pras40 [3], whereas Rictor, GβL, Protor, Deptor and mSin form mTORC2 [4,5]. mTORC1 regulates protein biosynthesis through phosphorylation of the ribsosomal protein S6 kinase (p70S6K) and subsequent phosphorylation of the ribosomal protein S6 (rpS6), or phosphorylation and inactivation of the eukaryotic initiation factor 4E (eIF4E)-binding protein-1(4E-BP1), dissociating 4E-BP1 from the RNA cap-binding protein eIF4E, thus promoting cap-dependent translation of mRNAs [6]. In contrast to mTORC1 complex, mTORC2 is currently poorly understood. Rictor is the important component of mTORC2 [4].To determine the role of mTORC1 in development, we studied the effects of deleting Rictor on embryogenesis.

Results

The Cre/loxP system was used to develop conditional Rictor KO mutants. A previously established mutant mouse line was used, n which Rictor was flanked by 2 loxP sequences. To obtain conditional Rictor KO, we then crossed mutant mice harboring the Rictor floxed allele with mice, which have Cre- recombination. to deleted of.

When expressing Cre recombinase started under the control of the K14 promoter the loxP-flanking region was deleted. Genotypes were confirmed by PCR of tail DNA, using oligonucleotide primers pairs for floxed Rictor and for the Cre transgene. The genotyping was confirmed by Western blot of the total lysates of the embryos epithelial cells to show that the Akt/PKB expression.

In these studies, we generated mice deficient for rictor in epithelial tissues to determine the role of mTORC2 in the embryonic development of mammals.

Immunoblot analyses of protein lysates prepared from rictor-deficient epithelial cells reveal that gene products are necessary for the basal phosphorylation of Akt/PKB on S473 of its hydrophobic motif. Rictor-deficient embryos die at early stages of development (8.5 days).

We conclude that rictor is an essential component of mTORC2 and is required in early development.

A B

Fig. 1 Phenotypic Characterization of Embryos (A) – 15.5 days, (B) – 8.5 days

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