V. V. Filonenko

Negative regulation of PI 3-kinase by Ruk, a novel adaptor protein.

Class IA phosphatidylinositol 3‐kinase (PI 3‐kinase) is a key component of important intracellular signalling cascades. We have identified an adaptor protein, Rukl, which forms complexes with the PI 3‐kinase holoenzyme in vitro and in vivo. This interaction involves the proline‐rich region of Ruk and the SH3 domain of the p85α regulatory subunit of the class IA PI 3‐kinase. In contrast to many other adaptor proteins that activate PI 3‐kinase, interaction with Rukl substantially inhibits the lipid kinase activity of the enzyme. Overexpression of Rukl in cultured primary neurons induces apoptosis, an effect that could be reversed by co‐expression of constitutively activated forms of the p110α catalytic subunit of PI 3‐kinase or its downstream effector PKB/Akt. Our data provide evidence for the existence of a negative regulator of the PI 3‐kinase signalling pathway that is essential for maintaining cellular homeostasis. Structural similarities between Ruk, CIN85 and CD2AP/CMS suggest that these proteins form a novel family of adaptor molecules that are involved in various intracellular signalling pathways.

Molecular cloning and characterization of a novel p70 S6 kinase, p70 S6 kinase beta containing a proline-rich region.

A novel ribosomal S6 kinase, termed p70 S6 kinase β (p70β), which has a highly conserved amino acid sequence compared with that of p70/p85 S6 kinase (p70α) within the catalytic, kinase extension, and autoinhibitory pseudosubstrate domains, was identified. However, the amino acid sequence of p70β differs from that of p70α in the noncatalytic amino-terminal region and in the carboxyl-terminal tail, which contains a proline-rich region. The majority of the regulatory phosphorylation sites identified in p70α are conserved in p70β. Two isoforms of p70β, referred to as β1 (495 amino acids) and β2 (482 amino acids), could be expressed from the single gene either by alternative mRNA splicing or by the use of alternative start codons. Here we report the characterization of p70β2. Similarly to p70α, the catalytic activity of p70β toward ribosomal protein S6 could be rapidly activated by serum, insulin, and phorbol ester in transiently transfected cells. The p70β kinase was found to be significantly less sensitive to wortmannin and rapamycin than p70α. These results indicate that p70β has the potential to participate in the regulation of protein synthesis and the cell cycle.