Phosphorylation of Ser72 is dispensable for Skp2 assembly into an active SCF ubiquitin ligase and its subcellular localization

Abstract

F-box proteins are the substrate recognition subunits of SCF (Skp1, Cul1, F-box protein) ubiquitin ligase complexes. Skp2 is a nuclear F-box protein that targets the CDK inhibitor p27 for ubiquitin- and proteasome-dependent degradation. In G(0) and during the G(1) phase of the cell cycle, Skp2 is degraded via the APC/C(Cdh1) ubiquitin ligase to allow stabilization of p27 and inhibition of CDKs, facilitating the maintenance of the G(0)/G(1) state. APC/C(Cdh1) binds Skp2 through an N-terminal domain (amino acids 46-94 in human Skp2). It has been shown that phosphorylation of Ser64 and Ser72 in this domain dissociates Skp2 from APC/C. More recently, it has instead been proposed that phosphorylation of Skp2 on Ser72 by Akt/PKB allows Skp2 binding to Skp1, promoting the assembly of an active SCF(Skp2) ubiquitin ligase, and Skp2 relocalization/retention into the cytoplasm, promoting cell migration via an unknown mechanism. According to these reports, a Skp2 mutant in which Ser72 is substituted with Ala is unable to promote cell proliferation and loses its oncogenic potential. Given the contrasting reports, we revisited these results and conclude that phosphorylation of Skp2 on Ser72 does not control Skp2 binding to Skp1 and Cul1, has no influence on SCF(Skp2) ubiquitin ligase activity, and does not affect the subcellular localization of Skp2.

Figure 1

Mutation of Ser72 does not affect the interaction of Skp2 with Skp1 or Cul1. HEK293T cells were transfected with the indicated FLAG-tagged Skp2 constructs or an empty vector (EV). Twenty-four hours post-transfection, whole cell extracts (WCE) were subjected to immunoprecipitation (IP) with anti-FLAG resin and immunoblotting was performed for the indicated proteins.

Figure 2

Pharmacologic inhibition of the PI(3)K-Akt pathway does not affect the binding of Skp2 to Skp1 or Cul1. HEK293T cells were stimulated with serum for 6 hours and treated with vehicle or 20 mM LY294002 for the final 5 hours. Endogenous Skp2 was immunoprecipitated, and SCF complex formation was monitored by immunoblotting with the indicated antibodies. The activation status of Akt1 was monitored by immunoblotting with anti-phospho-Akt1(Ser473) antibody. NIA, non-immunoprecipitating antibody.

Figure 3

Mutation of Ser72 does not influence the in vitro ubiquitylation activity of Skp2. One microgram of in vitro transcribed/translated [³⁵S]-labeled p27 produced in rabbit reticulocyte extract (which contains Skp1, Cul1 and Rbx1) was incubated in the presence of 0.1 μl purified, recombinant Cdk2-cyclin E complex and 0.1 μl purified, recombinant Cks1. In addition, the reaction was supplemented with 1 μl of in vitro transcribed/translated cold Skp2 (wild type and mutants), as indicated. The right bracket indicates a ladder of bands of relative molecular mass >27,000 Da, corresponding to polyubiquitylated p27; the arrow indicates unconjugated [³⁵S]p27.

Figure 4

Mutation of Ser72 does not influence the in vivo ubiquitylation activity of Skp2. HEK293T cells were transfected with His-tagged ubiquitin and either one of the indicated FLAG-tagged Skp2 constructs or an empty vector (EV). Twenty-four hours post-transfection, whole cell extracts (WCE) were treated with the proteasome inhibitor MG-132 for four hours prior to collection. After purification of ubiquitylated proteins with Ni-NTA resin, proteins were analyzed by immunoblotting with an antibody to p27. The bracket on the right side of the panels marks a ladder of bands >27,000 Da, corresponding to polyubiquitylated p27. The bottom panel shows the level of exogenous Skp2 as detected by immunoblotting.

Figure 5

Mutation of Ser72 in Skp2 does not influence the cellular localization of Skp2. U-2 OS cells were transfected with the indicated FLAG-tagged Skp2 constructs in the absence or presence of HA-tagged myr-Akt1, and 48 hours post-transfection, cells were collected and analyzed by immunofluorescence with an anti-FLAG antibody, DAPI and anti-HA antibody, as indicated.