The Tumor Suppressor Protein p16INK4a and the Human Papillomavirus Oncoprotein-58 E7 Are Naturally Occurring Lysine-less Proteins That Are Degraded by the Ubiquitin System: DIRECT EVIDENCE FOR UBIQUITINATION AT THE N-TERMINAL RESIDUE

Ronen Ben-Saadon,Ifat Fajerman,Tamar Ziv,Ulf Hellman,Alan L Schwartz,Aaron Ciechanover

doi:10.1074/jbc.m407201200

Abstract

Conjugation of ubiquitin to an internal lysine is the initial step in the degradation of the majority of the substrates of the ubiquitin system. For several substrates, it has been shown that the first ubiquitin moiety is conjugated to the N-terminal residue. In all these substrates, however, the internal lysines also played a role in modulating their stability. To better understand the physiological significance of this novel mode of modification, it was important to identify proteins in which degradation is completely dependent on N-terminal ubiquitination. Also, although the experimental evidence for N-terminal ubiquitination is rather strong, nevertheless, it has remained indirect. Here we demonstrate that an important group of proteins that are targeted via N-terminal ubiquitination are the naturally occurring lysine-less proteins such as the human papillomavirus (HPV)-58 E7 oncoprotein and the cell cycle inhibitor and tumor suppressor p16(INK4a). For these proteins, the only residue that can be targeted is the N-terminal residue. Interestingly, p16(INK4a) is degraded in a cell density-dependent manner. Importantly, we provide for the first time direct evidence for N-terminal ubiquitination. Analysis of tryptic digest of the ubiquitin conjugate of HPV-58 E7 revealed a fusion peptide that is composed of the C-terminal domain of ubiquitin and the N-terminal domain of E7. With the abundance of native lysine-less proteins, among which are important viral and cell regulators, this novel mode of protein targeting has implications for both physiological and pathophysiological processes.

Highlights

Conjugation of ubiquitin to an internal lysine is the initial step in the degradation of the majority of the substrates of the ubiquitin system
We demonstrate that an important group of proteins that are targeted via N-terminal ubiquitination are the naturally occurring lysine-less proteins such as the human papillomavirus (HPV)-58 E7 oncoprotein and the cell cycle inhibitor and tumor suppressor p16INK4a
The naturally occurring lysine-less proteins (NOLLPs) HPV-58 E7 Is Targeted by the Ubiquitin-Proteasome System in Vitro and in Vivo—Once HPV-58 E7 had been identified as a NOLLP (Fig. 1A), it was important to examine whether it is degraded by the ubiquitin system

Summary

Introduction

Conjugation of ubiquitin to an internal lysine is the initial step in the degradation of the majority of the substrates of the ubiquitin system. We demonstrate that an important group of proteins that are targeted via N-terminal ubiquitination are the naturally occurring lysine-less proteins such as the human papillomavirus (HPV)-58 E7 oncoprotein and the cell cycle inhibitor and tumor suppressor p16INK4a. N-terminal tagging probably stabilizes MyoD by blocking access of ubiquitin to the specific ubiquitination site at the N terminus; it can affect recognition of the substrate by the E3 by altering the structure of the N-terminal domain. Taken together, these results strongly suggest that the first event in ubiquitin-mediated degradation of MyoD involves fusion of ubiquitin to the N-terminal residue. Additional ubiquitin moieties are added to Lys of the previously conjugated moiety [3]

Methods

Results

Conclusion