Cell cycle-dependent caspase-like activity that cleaves p27 KIP1 is the b 1 subunit of the 20S proteasome Winston S. Tambyrajah a , Lucas D. Bowler b , Cahora Medina-Palazon a , Alison. J. Sinclair a, * a School of Life Sciences, University of Sussex, Brighton BN1 9QG, UK b University of Sussex Proteomics Facility, University of Sussex, Brighton BN1 9RY, UK Received 22 June 2007, and in revised form 13 July 2007 Available online 6 August 2007 Abstract We previously described a caspase-like activity, which we termed KIPase that is implicated in the turnover of the mammalian cell cycle regulator p27 KIP1 . KIPase cleaves a tetra-peptide substrate, Ac-DPSD-AMC, which mimics the target site in p27 KIP1 , and inhib- itors based on this tetra-peptide are ineffective against other known caspases. Here we describe the purification and characterization of KIPase, and trace its activity to the b 1 subunit of the 20S proteasome. Further analyses revealed that the activity of the b 1 subunit is up- regulated as cells enter the cell cycle without concomitant change in the levels of the proteasome b 1 , b 2 or b 5 subunits. To our knowledge, this is the first description of cell cycle regulation of the caspase-like activity of the 20S proteasome. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Proteasome; Cell cycle; Caspase; p27 KIP1 ; Lymphoid cells Intracellular protein degradation is an essential and tightly regulated process in all living cells. Proteasomes are responsible for the degradation of the majority of pro- teins in a non-lysosomal manner [1]. The 26S proteasome, a large multi-subunit catalytic complex [2–9], is the major component of this pathway and causes the degradation of ubiquitylated substrates in an ATP-dependent manner [10–12]. The two inner b-rings each contain seven subunits [2–5] that confer three distinct proteolytic activities: a post- acidic or caspase-like activity, a trypsin-like activity and a chymotrypsin-like activity [13–16]. These activities are also present in the 20S proteasome, which targets denatured, but non-ubiquitylated proteins while a further variant, the immuno-proteasome is involved in the production of peptides required for antigen presentation by the major his- tocompatibility complex [17]. In mammalian cells, cell cycle re-entry from quies- cence is orchestrated by the balance between cyclin- dependent kinases (Cdks) and various Cdk inhibitors [18]. One such inhibitor, p27 KIP1 , is present at high levels in resting cells, but is degraded during the G1 to S phase transition [19]. Elevated levels of cyclins or reduced levels of p27 KIP1 have been reported in a number of human tumors and are associated with poor prognosis. Turnover of p27 KIP1 is in part triggered by Cdk-mediated phos- phorylation and ubiquitylation by the SKP2 complex [19–22]. However, we have previously shown that in pro- liferating lymphocytes, p27 KIP1 is specifically cleaved by a caspase-like activity that we have termed KIPase [23]. KIPase cuts p27 KIP1 at the tetra-peptide sequence DPSD, which differs subtly from the target sites of other known caspases. Indeed, inclusion of a proline at position P3 has been shown to generate a poor substrate for most caspases [24,25]. Importantly, a KIPase inhibitor, based on the DPSD tetra peptide, is unable to block the func- tion of known caspases [26]. 0003-9861/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2007.07.019 * Corresponding author. Fax: +44 1273 678 433. E-mail address: a.j.sinclair@sussex.ac.uk (A.J. Sinclair). www.elsevier.com/locate/yabbi Archives of Biochemistry and Biophysics 466 (2007) 186–193 ABB