Targeting of the Akt/PKB kinase to the actin skeleton.
Serine/threonine kinase Akt/PKB intracellular distribution undergoes rapid changes in response to agonists such as Platelet-derived growth factor (PDGF) or Insulin-like growth factor (IGF). The concept has recently emerged that Akt subcellular movements are facilitated by interaction with nonsubstrate ligands. Here we show that Akt is bound to the actin skeleton in in situ cytoskeletal matrix preparations from PDGF-treated Saos2 cells, suggesting an interaction between the two proteins. Indeed, by immunoprecipitation and subcellular fractioning, we demonstrate that endogenous Akt and actin physically interact. Using recombinant proteins in in vitro binding and overlay assays, we further demonstrate that Akt interacts with actin directly. Expression of Akt mutants strongly indicates that the N-terminal PH domain of Akt mediates this interaction. More important, we show that the partition between actin bound and unbound Akt is not constant, but is modulated by growth factor stimulation. In fact, PDGF treatment of serum-starved cells triggers an increase in the amount of Akt associated with the actin skeleton, concomitant with an increase in Akt phosphorylation. Conversely, expression of an Akt mutant in which both Ser473 and Thr308 have been mutated to alanine completely abrogates PDGF-induced binding. The small GTPases Rac1 and Cdc42 seem to facilitate actin binding, possibly increasing Akt phosphorylation.
Cenni
V
V
Laboratory of Cell Biology and Electron Microscopy, Rizzoli Orthopedic Institute, via di Barbiano 1/10, 40136 Bologna, Italy.
Sirri
A
A
Riccio
M
M
Lattanzi
G
G
Santi
S
S
de Pol
A
A
Maraldi
N M
NM
Marmiroli
S
S
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000199:Actins / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism
D000199:Actins; D036341:Intercellular Signaling Peptides and Proteins; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; C065700:myotrophin; D064413:3-Phosphoinositide-Dependent Protein Kinases; C494918:AKT1 protein, human; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020764:cdc42 GTP-Binding Protein