The N-terminal 34 residues of the 55 kDa regulatory subunits of phosphoinositide 3-kinase interact with tubulin

Kouichi Inukai, Makoto Funaki, Masao Nawano, Hideki Katagiri, Takehide Ogihara, Motonobu Anai, Yukiko Onishi, Hideyuki Sakoda, Hiraku Ono, Yasushi Fukushima, Masatoshi Kikuchi, Yoshitomo Oka, Tomoichiro Asano

Research output: Contribution to journalArticlepeer-review

35 Citations (Scopus)

Abstract

There are five regulatory subunit isoforms of phosphoinositide 3-kinase (PI 3-kinase), which are classified into three groups: proteins of 85 kDa (p85α and p85β), 55 kDa (p55α and p55γ) and 50 kDa (p50α). Structural differences between the three groups reside in the N-terminus. To elucidate the unique functional role of the 55 kDa regulatory subunits, GST (glutathione S-transferase) fusion proteins containing a unique N-terminal portion consisting of a 34-amino-acid sequence of p55α or p55γ (GST-p55α/γN1-34) were used as affinity matrices to screen rat brain cell extracts for proteins to which this portion binds specifically. A protein that bound was identified as β-tubulin by protein sequencing. In addition, not only the β isoform of tubulin, but also the α and γ isoforms, were detected in the protein absorbed from cell lysates with GST-p55γN1-34 and GST-p55αN1-34 by immunoblotting. Indeed, the only regulatory subunit present in the purified microtubule assembly from rat brain was the 55 kDa isoform; neither 85 kDa nor 50 kDa subunits were detected. These results indicate endogenous binding of 55 kDa regulatory subunits of PI 3-kinase to tubulin in the brain. Finally, we measured tubulin-associated PI 3-kinase activity in CHO/IR cells overexpressing each of the five regulatory subunit isoforms. Only in cells expressing p55α or p55γ was there a significant elevation of tubulin-associated PI 3-kinase activity in response to insulin. These results suggest that the p55α and p55γ regulatory subunits have important roles in regulating PI 3-kinase activity, particularly for microtubules at the cell periphery.

Original languageEnglish
Pages (from-to)483-489
Number of pages7
JournalBiochemical Journal
Volume346
Issue number2
DOIs
Publication statusPublished - 2000 Mar 1

Keywords

  • Insulin
  • Microtubules
  • PI 3-kinase
  • p55

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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