The N-terminal 34 residues of the 55 kDa regulatory subunits of phosphoinositide 3-kinase interact with tubulin.
There are five regulatory subunit isoforms of phosphoinositide 3-kinase (PI 3-kinase), which are classified into three groups: proteins of 85 kDa (p85alpha and p85beta), 55 kDa (p55alpha and p55gamma) and 50 kDa (p50alpha). 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 p55alpha or p55gamma (GST-p55alpha/gammaN(1-34)) 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 beta-tubulin by protein sequencing. In addition, not only the beta isoform of tubulin, but also the alpha and gamma isoforms, were detected in the protein absorbed from cell lysates with GST-p55gammaN(1-34) and GST-p55alphaN(1-34) 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 p55alpha or p55gamma was there a significant elevation of tubulin-associated PI 3-kinase activity in response to insulin. These results suggest that the p55alpha and p55gamma regulatory subunits have important roles in regulating PI 3-kinase activity, particularly for microtubules at the cell periphery.
Inukai
K
K
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Funaki
M
M
Nawano
M
M
Katagiri
H
H
Ogihara
T
T
Anai
M
M
Onishi
Y
Y
Sakoda
H
H
Ono
H
H
Fukushima
Y
Y
Kikuchi
M
M
Oka
Y
Y
Asano
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*
D010446:Peptide Fragments; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D019869:Phosphatidylinositol 3-Kinases