Characterization of the Akt2 Domain Essential for Binding Nuclear p21cip1 to Promote Cell Cycle Arrest during Myogenic Differentiation

Lamb NJ (2013) Characterization of the Akt2 Domain Essential for Binding Nuclear p21cip1 to Promote Cell Cycle Arrest during Myogenic Differentiation. PLoS ONE 8(10): e76987. doi:10.1371/journal.pone.0076987 Characterization of the Akt2 Domain Essential for Binding Nuclear p21cip1 to Promote Cell Cycle Arrest during Myogenic Differentiation Lisa Heron-Milhavet 0 Celine Franckhauser 0 Anne Fernandez 0 Ned J. Lamb 0 Imed Eddine Gallouzi, McGill University, Canada 0 Cell Cycle and Myogenesis, Institute of Human Genetics , CNRS-UPR1142, Montpellier , France 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. - Funding: This work was supported by ARC grant #3976 (http://www.arc-cancer.net/) and LNCC Grant (Herault et Aude) (http://www.ligue-cancer.net). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The serine-threonine kinase Akt was first discovered as the oncogene in the transforming retrovirus AKT8 [1] and it has become the subject of intense research since then because of its implication in cancer progression, metabolism, cellular growth and differentiation, and survival. Three isoforms of Akt have been identified: Akt1, Akt2 and Akt3 and their tissue distribution has been determined [2] showing that both Akt1 and Akt2 isoforms are ubiquitously expressed, whereas the Akt3 isoform is not detected in several tissues where Akt1 and Akt2 are highly expressed, but is relatively highly expressed in brain and in testis. Akt2 is expressed predominantly in insulin target tissues, such as fat cells, liver and skeletal muscle. The three Akt isoforms possess the kinase domain in the central region of the molecule; the PH (pleckstrin homology) domain acts as phosphoinositide-binding molecule and the hydrophobic motif (HM) is located at the carboxy-terminal adjacent to the kinase domain [3]. Akt is activated by a multistep process that results in phosphorylation of two critical residues threonine 308 in the activation loop and serine 473 in the hydrophobic motif, which induces a substantial conformational change that leads to a greater than 1000-fold increase in its kinase activity [46]. The initiation step in the activation of Akt is its recruitment to the plasma membrane where the PH domain directs the translocation of Akt from the cytosol to the plasma membrane by binding to the products of PI3K. We have published in 2006 and in 2008 the respective role of Akt1 and Akt2 isoforms in the regulation of cell cycle proliferation and exit towards myogenic differentiation. We have shown that Akt1 is implicated in cell cycle progression whereas Akt2, principally through its interaction with the cdk inhibitor p21cip1, is implicated in cell cycle exit thus promoting myoblast differentiation [78]. In this study, we have determined the region on Akt2 necessary for the binding with p21cip1 is a 27 amino acid sequence spanning the C-terminal region 410437 of Akt2, showing strong differences with Akt1 in both primary sequence and secondary structure. Results and Discussion We have previously shown that Akt2 interacts with p21cip1, inducing its stabilization in the nucleus promoting cell cycle arrest and entry into myogenic differentiation [7]. In the present study, we have cloned, produced and purified various forms of human Akt2 proteins truncated specifically at the N-terminal and Cterminal Akt2 regions (see Figure S1 for details) as well as Akt2derived peptides to study the binding site between Akt2 and p21. We first examined whether p21cip1 bound to Akt2 through its amino or carboxy terminal region. Three N-terminal deletions of Akt2 were expressed in bacteria with C-terminal 6 His fusion tag and after purification, these deletion proteins were probed for the capacity to bind p21 by pull down assay using anti-p21. As shown in figure 1A, deletion of amino acids 1350 or 1400 did not affect the capacity of Akt2 to bind p21. In contrast, a truncated form of Akt2 in which only the amino acids 430481 remained was unable to bind p21 in vitro. To confirm the importance of the C-terminal region of Akt2 in the binding to p21, we next performed the reciprocal experiment, truncating regions from the C-terminus. As shown in figure 1B, truncation of amino acids 460481 and 445 481 had no effect on the interaction with p21 whereas a truncation spanning amino acids 430481 was no longer capable of binding p21. These data suggest that a region between amino acids 400 445 of Akt2 is necessary for the binding to p21 in vitro. This region corresponds to the site at which the primary sequences of Akt1 and Akt2 diverge the most, consistent with being potentially the sequence through which Akt2 binds specifically p21cip1, an interaction never observed with Akt1 [7]. We next examined if we could reduce this region by producing Akt2 peptides as maltose binding fusion proteins (MBP). This has the dual advantage that the interactions observed above occur through the presence of 6 Hi sequences in both p21 and Akt2 truncations and that the complexes formed can be extensively washed to reinforce the robustness of the binding. As shown in figure 1C, when p21cip1 was incubated with either Akt2 (400445) or Akt2 (420445), there was a clear binding to both truncation peptides. The binding between Akt2 (400445) and p21cip1 was even slightly less efficient than that of Akt2 (420445) since it showed no increase in p21 binding when the levels of (...truncated)