Targeting p21 Degradation Locally

Abstract

Just as the activity of many multifunctional proteins is restricted by subcellular localization, so is their regulation. In this issue of Development Cell, Starostina et al. identify an E3 ubiquitin ligase, CRL2^LRR1, for the cyclin-dependent kinase inhibitor p21 that specifically ubiquitylates cytoplasmic p21 to facilitate cell migration.

The ubiquitin system covalently attaches ubiquitin to substrate proteins, leading to either a nonproteolytic change of function or degradation by the 26S proteosome. While a limited number of E1-activating and E2-conjugating enzymes are employed by the system, more than a thousand distinct E3 ubiquitin ligases are present in higher eukaryotes to regulate a wide range of cellular process. Proteasomes and E3 ligases are present in both the nucleus and the cytosol, and ubiquitin-mediated degradation occurs in both compartments. There have been, however, relatively few examples of localized ubiquitylation reported thus far. Two notable examples are p53 and b-catenin, transcription factors that can be exported out of the nucleus for degradation in the cytoplasm. This nuclear export and cytoplasmic ubiquitylation ensures that the function of the transcription factor is geographically separated from its degradation to avoid promiscuous degradation and to allow rapid activation of the factor in response to signaling events by simply blocking export and ubiquitylation. Reporting in this issue of Developmental Cell, Starostina and colleagues (2010) now identify a specific E3 ligase for the degradation of cytoplasmic p21 in human cells, providing evidence that distinct functions of an individual protein in different cellular compartments can be regulated by different E3 ligases.

At the heart of this study is p21^CIP1/WAF1, best known as a transcriptional target of p53 following DNA damage and other cellular stresses. p21 induces a G1 cell-cycle arrest by inhibiting cyclin-dependent kinases (CDKs) in the nucleus. Additional functions for p21 and its cousin p27^KIP1 were uncovered by a number of studies reporting cytoplasmic accumulation of the proteins in multiple types of human tumors, suggesting that p21 and p27 could function as tumor suppressors in the nucleus and oncoproteins in the cytoplasm (Besson et al., 2004). The cytoplasmic function of p21 is not dependent on its ability to inhibit CDKs but rather is linked to actin cytoskeleton regulation. Cytoplasmic p21 inhibits the Rho-associated kinase, ROCK1, uncoupling Rho-GTPase activity from stress fiber formation and promoting cytoskeleton remodeling and cell motility. Are these distinctive nuclear and cytoplasmic functions of p21 differentially regulated? The study by Starostina et al. (2010) provides compelling evidence supporting just such a model.

In a search for proteins that interact with Caenorhabditis elegans CUL2, a member of evolutionary conserved cullin family proteins that function as scaffold for the assembly of cullin-RING E3 ligases (CRLs), Starostina et al. (2010) identified by affinity purification the leucine-rich repeat protein 1 (LRR1), a VHL-box protein brought to the CUL2 complex via its interaction with the CUL2 linker protein, elongin C (Figure 1). The authors previously showed that in C. elegans, cul-2 mutants exhibit a germline defect, with germ cells arrested in G1 (Feng et al., 1999). This defect could be partially rescued by the haploid deletion of cki-1, the C. elegans homolog of mammalian p27, suggesting that it is a downstream effector of CUL2 function. Starostina and colleagues (2010) observed that mutation of lrr-1 was phenotypically similar to cul-2 mutation, resulting in accumulation of CKI-1. Likewise, the G1 arrest defect of germ cells in lrr-1 mutants was partially rescued by deletion of one cki-1 allele. Furthermore, the authors found that LRR1 physically binds to CKI-1, and its overexpression increased CKI-1 protein turnover. Collectively, these genetic and biochemical data are consistent with the notion that in C. elegans, LRR-1 functions to promote the G1 to S progression by serving as substrate receptor to target CKI-1 for ubiquitylation by the CRL2^LRR1 E3 ligase (Figure 1).

Figure 1. Multiple E3 ligases target human p21 ubiquitylation in different cellular compartments.

Four E3 ligases—SCF^SKP2, APC/C^CDC20, CRL4^CDT2, and CRL2^LRR1—are involved in p21 ubiquitylation and destruction. Unlike the three previously identified E3 ligases, which act on p21 in the nucleus in the context of cell-cycle regulation, CRL2^LRR1 targets p21 in the cytoplasm to control cell migration. All four E3 ligases are all composed of multiple subunits, including a scaffold (green) that binds to a small RNG finger protein, ROC1/RBX1 or APC11 (red), which in turn brings in and allosterically activates a ubiquitin (Ub)-conjugating enzyme, E2 (brown). A separate domain in the scaffold binds to a linker protein (blue) that interacts with a protein motif (orange) present in substrate receptors (black).

The authors then investigated the function of mammalian LRR1 and found that knocking down LRR1 selectively increased the protein level of p21 but not the related CDK inhibitors p27 and p57. Surprisingly, unlike in C. elegans cells, depletion of either LRR1 or CUL2 in human cells did not cause an obvious cell cycle arrest. This unexpected finding led the authors to examine CRL2^LRR1 activity specifically in cytoplasm, where p21 functions to regulate ROCK1 independent of its role in cell-cycle regulation. They found that cytoplasmic p21 did indeed accumulate in response to knockdown of either LRR1 or CUL2. Furthermore, LRR1 or CUL2 knockdown decreased stress fibers and cell-cell contacts while increasing F-actin at the cell periphery and cell motility, consistent with a role for the protein in regulating cytoplasmic p21. Importantly, these morphological changes in LRR1-knockdown cells were suppressed by codepletion of p21.

Three different E3 ubiquitin ligases have been previously identified in targeting p21 degradation (Figure 1): SCF^SKP2 (Bornstein et al., 2003; Wang et al., 2005; Yu et al., 1998), APC/C^CDC20 (Amador et al., 2007), and CRL4^CDT2 (Abbas et al., 2008; Kim et al., 2008; Nishitani et al., 2008). Given that SKP2, CDC20, and CDT2—the substrate recognition subunits for the respective E3 ligase complexes—are all nuclear proteins, these E3s probably ubiquitylate only nuclear p21. No evidence links these three E3 ligases with either the ubiquitylation of cytoplasmic p21 or the regulation of cytoskeleton and cell motility, making the CRL2^LRR1 the first E3 ligase specific for cytoplasmic p21.

The study by Starostina et al. (2010) raises several interesting questions. Despite the important function of CRL2^LRR1 in regulating cytoskeleton dynamics and cell migration, this function is not conserved in C. elegans. No obvious cell migration defect was detected by the authors in C. elegans lrr-1 mutants. Moreover, C. elegans LRR1 is localized to the nucleus. How broadly does the CRL2^LRR1 E3 ligase target cytoplasmic p21? Did CRL2^LRR1-mediated cytoplasmic p21 ubiquitylation evolve after the acquisition of the cytoplasmic CIP/KIP function that is restricted to mammalian cells? Targeting the ubiquitylation of an individual protein with multiple E3 ligases is not unique to p21—at least three E3 ligases (SCF^SKP2, KPC, and CRL4) are involved in p27 ubiquitylation, and more than a dozen are linked to p53 ubiquitylation. So how do cells coordinate between different E3 ligases, especially when the physiological outcomes of this differential targeting are distinct? The answer probably rests in the upstream trigger that leads to the binding of the substrate to its receptor, an issue that has not been addressed by the current work but the analysis of which is an important next step in understanding the pathways that orchestrate p21’s varied functions.

The finding by Starostina et al. (2010) of targeted protein degradation in a specific subcellular compartment also suggests that the function of some E3 ligases may escape detection by commonly used techniques such as western blotting of whole-cell or tissue lysates. Subcellular fractionation or immunohistochemistry may be needed for studying substrates or recognition factors that are known to localize to a specific compartment. Lastly, the work of Starostina et al. (2010) may have implications for tumorigenesis. LRR1 has thus far not been well characterized, but, as the authors note, it is located on human chromosome 14q21.3, a region that is lost in several types of metastatic tumors. Given that cytoplasmic accumulation of p21 facilitates cell migration and potentially tumor metastasis, one wonders whether LRR1 functions to suppress late stages of tumorigenesis.