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.
The N-terminal 34 residues of the 55 kDa regulatory subunits of phosphoinositide 3-kinase interact with tubulin
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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; The N-terminal 34 residues of the 55 kDa regulatory subunits of phosphoinositide 3-kinase interact with tubulin. Biochem J 1 March 2000; 346 (2): 483–489. doi: https://doi.org/10.1042/bj3460483
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