TIP120A Associates with Cullins and Modulates Ubiquitin Ligase Activity

Abstract

The cullin-containing ubiquitin-protein isopeptide ligases (E3s) play an important role in regulating the abundance of key proteins involved in cellular processes such as cell cycle and cytokine signaling. They have multisubunit modular structures in which substrate recognition and the catalysis of ubiquitination are carried out by distinct polypeptides. In a search for proteins involved in regulation of cullin-containing E3 ubiquitin ligases we immunopurified CUL4B-containing complex from HeLa cells and identified TIP120A as an associated protein by mass spectrometry. Immunoprecipitation of cullins revealed that all cullins tested specifically interacted with TIP120A. Reciprocal immunoaffinity purification of TIP120A confirmed the stable interaction of TIP120A with cullin family proteins. TIP120A formed a complex with CUL1 and Rbx1, but interfered with the binding of Skp1 and F-box proteins to CUL1. TIP120A greatly reduced the ubiquitination of phosphorylated I B␣ by SCF ␤-TrCP ubiquitin ligase. These results suggest that TIP120A functions as a negative regulator of SCF E3 ubiquitin ligases and may modulate other cullin ligases in a similar fashion.The ubiquitin-dependent proteolysis provides a fundamental mechanism for regulating protein activity in various processes ranging from cell cycle and developmental switches to signal transduction (1). This process begins with the attachment of a multiubiquitin chain to a target protein and involves several enzymatic activities. A ubiquitin-activating enzyme (E1) 1 activates ubiquitin in an ATP-dependent reaction by forming a thioester bond with the C-terminal glycine of ubiquitin. The ubiquitin is then transferred to a specific sulfhydryl group on a ubiquitin-conjugating enzyme (E2). A ubiquitin-protein ligase (E3) transfers the activated ubiquitin from E2 to a lysine residue of a bound substrate, forming an isopeptide bond. Substrate specificity is determined mainly by E3s which bind both the protein substrate and the cognate E2. Once the multiubiquitin chain is assembled on a protein substrate by the cooperation of E1, E2, and E3 enzymes, the target protein is recognized and degraded by the 26 S proteasome (1-3).In mammalian cells, a wide variety of E3s are found. The cullin family proteins play an important role in a group of multisubunit E3 ubiquitin ligases by associating with an Rbx1 (also known as ROC1 and Hrt1) family member of RING finger proteins to form the integral core (4). The SCF complexes are the best characterized ones of this class (5). They consist of CUL1, Rbx1, Skp1, and an F-box protein. Rbx1 contains the RING-H2 finger domain, forms a catalytic core with CUL1, and recruits the cognate E2 (6 -8). Skp1 functions as an adaptor that links an F-box protein to CUL1 (9). Substrates of the SCF complexes are bound by F-box proteins, which contain the Skp1-binding F-box motif and a variable protein-protein interaction domain that directly interacts with substrates (9, 10). Since a large number of F-box proteins are encoded by eukaryotic genomes (11-13), ...

Nedd8 on cullin: building an expressway to protein destruction

“…Nedd8 conjugation reverses p120 CAND1 -mediated inhibition p120 CAND1 was found to selectively interact with unneddylated CUL1 and ROC1/Rbx1 (Liu et al, 2002;Zheng J. et al, 2002;Min et al, 2003;Oshikawa et al, 2003). Moreover, this interaction appeared to cause dissociation of Skp1 from CUL1, hence resulting in inhibition of SCF E3 ligase activity.…”

Selenoprotein S-dependent Selenoprotein K Binding to p97(VCP) Protein Is Essential for Endoplasmic Reticulum-associated Degradation

“…B. Yoon (Yonsei University, Seoul, Korea). The pYR-CD3␦-FLAG construct contains a tetracycline-regulated promoter (18,29). The pYR-CD3␦-FLAG and pTet-off (Clontech) plasmids, which encodes a tetracycline-controlled transactivator, were co-transfected into N2a cells to express CD3␦-FLAG.…”

CAND1-Mediated Substrate Adaptor Recycling Is Required for Efficient Repression of Nrf2 by Keap1

“…A recent crystal structure of the CAND1-Cul1 complex, which showed that Lys 720 in Cul1 is located at the CAND1:Cul1 interface, has provided support for the notion that neddylation of Cul1 and binding of CAND1 are antagonistic (17). However, it is not clear if CAND1 is a global regulator of all cullin-dependent ubiquitin ligase complexes (28,30,45).…”

“…The second step is the association of a protein known as CAND1 (also termed TIP120A) with the deneddylated cullin protein (17,28,45). CAND1 binds to both N-terminal and C-terminal sequences in Cul1 and blocks binding of the substrate adaptor protein (17,19,28,30,45). Subsequent conjugation of Nedd8 onto the cullin subunit by Ubc12, a Nedd8-specific E2 enzyme (5), is proposed to decrease the affinity of CAND1 for the cullin protein, enabling another substrate adaptor protein (presumably with its bound substrate) to displace CAND1 and initiate another cycle of substrate ubiquitination (5,35).…”