title: Characterization of the Akt2 domain essential for binding nuclear p21cip1 to promote cell cycle arrest during myogenic differentiation.
abstract: The binding of the cdk inhibitor p21cip1 to Akt2 in the nucleus is an essential component in determining the specific role of Akt2 in the cell cycle arrest that precedes myogenic differentiation. Here, through a combination of biochemical and cell biology approaches, we have addressed the molecular basis of this binding. Using amino-terminal truncation of Akt2, we show that p21cip1 binds at the carboxy terminal of Akt2 since deletion of the first 400 amino acids did not affect the interaction between Akt2 and p21cip1. Pull down using carboxy terminal-truncated Akt2 protein revealed the importance of the region between amino acids 400 and 445 for the binding to p21cip1. Since Akt2_400-445 and Akt2_420-445 peptides could both bind p21cip1, this refines the binding domain on Akt2 between amino acids 420 and 445. In order to confirm these data in living cells, we developed a protocol to synchronize myoblasts at the cell cycle exit point when p21cip1 expression is induced by MyoD before myogenic differentiation. When a synthetic Akt2 peptide spanning the region (410-437) was microinjected in p21-expressing myoblasts, p21cip1 no longer localized exclusively in the nucleus, instead being redistributed throughout the cell, thus showing that injected peptide 410-437 acts to compete with the binding of endogenous Akt2 to p21cip1. Taken together, our data suggest that this 27 amino acid sequence on Akt2 is necessary and sufficient to bind p21cip1 both in vitro and in living cells.
authors.affiliation: Cell Cycle and Myogenesis, Institute of Human Genetics, CNRS-UPR1142, Montpellier, France.
authors.forename: Lisa
authors.initials: L
authors.lastname: Heron-Milhavet
authors.forename: Celine
authors.initials: C
authors.lastname: Franckhauser
authors.forename: Anne
authors.initials: A
authors.lastname: Fernandez
authors.forename: Ned J
authors.initials: NJ
authors.lastname: Lamb
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D059447:Cell Cycle Checkpoints / Q000235:genetics / Q000502:physiology*; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D024510:Muscle Development / Q000235:genetics / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics* / Q000378:metabolism; D017385:Sequence Homology
chemical_list: C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; C494922:Akt2 protein, mouse; D051057:Proto-Oncogene Proteins c-akt