Smurf1 ubiquitin ligase targets Kruppel-like factor KLF2 for ubiquitination and degradation in human lung cancer H1299 cells

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Abstract

Krüppel-like factor 2 (KLF2) has been demonstrated to be essential for normal lung development, erythroid differentiation, T-cell differentiation, migration and homing. However, the mechanisms underlying the regulation of KLF2, in particular its responsible E3 ligase is still unclear. Here we show that the homologous to E6AP carboxyl terminus (HECT)-type ubiquitin ligase Smad ubiquitination regulatory factor 1 (Smurf1) interacts with and targets KLF2 for poly-ubiquitination and proteasomal degradation specifically in lung cancer H1299 cells. The catalytic ligase activity of Smurf1 is required for it to regulate KLF2. Consequently, Smurf1 represses the transcriptional factor activity of KLF2 and regulates the expression its downstream genes such as CD62L and Wee1. This study provided the first evidence that Smurf1 functions as an E3 ligase to promote the ubiquitination and proteasomal degradation of KLF2.

Highlights

► The HECT-type ubiquitin ligase Smurf1 interacts with and targets KLF2 for poly-ubiquitination and proteasomal degradation specifically in lung cancer cells. ► Smurf1 represses the transcriptional factor activity of KLF2 and regulates the expression its downstream genes such as CD62L and Wee1. ► This study provided the first evidence that Smurf1 functions as an E3 ligase to promote the ubiquitination and proteasomal degradation of KLF2.

Introduction

Krüppel-like factors are a subclass of the zinc-finger family of transcription factors implicated in the regulation of cellular growth and differentiation. Since the initial discovery of EKLF in 1993, a total of 17 mammalian Krüppels have been identified and designated based on the chronological order of discovery (i.e., KLF1–17) [1], [2]. Human KLF2, owing to its high expression in lung tissues, was initially termed lung KLF (LKLF), and KLF2 has been demonstrated to be essential for normal lung development. The KLF2 knock-out mice exhibit impaired blood vessel formation attributable to the lack of smooth muscle cell recruitment [3], [4]. KLF2 can function as either a transactivator or repressor [5]. For example, KLF2 has been found to upregulate the expression of G1/S checkpoint gene p21 to inhibit leukemia cell growth [6]. On the contrary, KLF2 inhibits the expression of G2/M tyrosine kinase Wee1 to cause DNA damage-induced apoptosis [7]. Additionally, KLF2 is known to regulate the signaling pathways involved in thymocyte and T-cell trafficking. In this regard, KLF2 directly activates the promoters of CD62L and sphingosine-1-phosphate receptor 1, whose expression is critical for T cell egress from the thymus and homing to the lymph nodes [8].

Although the functions and the downstream targets have been widely studied, less is known about the stability control of KLF2 protein. Previous studies have indicated that KLF2 protein can be targeted to proteasome system for degradation. Although the HECT-type ubiquitin ligase WWP1 (WW domain-containing protein 1) was shown to promote the ubiquitination and degradation of KLF2, the catalytic activity of WWP1 enzyme seems to be not required for this degradation [9]. Therefore, one could speculate that WWP1 may not function as a direct E3 ligase for KLF2. At present, the identity of the ligase for KLF2 still remains unclear.

Here we provide evidence to show that Smad ubiquitination regulatory factor 1 (Smurf1) interacts with KLF2 both in vivo and in vitro, and directly targets the ubiquitination and proteasomal degradation of KLF2 specifically in lung cancer H1299 cells. Both Smurf1 and WWP1 belong to the Nedd4 family of HECT-type ligases and contain similar C2-WW-HECT structure. Smurf1 has been demonstrated to play a pivotal role in control of cell polarity, maintenance of bone homeostasis and regulation of tumorigenesis through targeting BMP-Smad, RhoA signaling pathways [10], [11]. The current study adds KLF2 to the substrate list of Smurf1 and establishes the functional relationship between Smurf1 and KLF2-mediated transcriptional control.

Section snippets

Plasmid constructs, antibodies and reagents

Full-length and truncated forms of KLF2 and Smurf1 were constructed by PCR, followed by subcloning into various vectors. Anti-Myc antibody was from Clontech. Anti-Flag M2 monoclonal antibody, the protein synthesis inhibitor cycloheximide (CHX) and the proteasome inhibitor MG132 were from Sigma. Anti-KLF2 polyclonal antibody (ab-28526) was from Abcam. Anti-GFP antibody was from Cell Signaling Technology and anti-HA antibody was from Roche. Anti-GST and his antibodies were from Tiangen. GAPDH and

Smurf1 interacts with KLF2 both in vitro and in vivo

The WW domains of Nedd4 family of ubiquitin ligases are usually responsible for substrate recognition. In a yeast two-hybrid screen with the WW domains of human Smurf1 as bait in a human brain cDNA library, we identified the full length of the KLF2 as an interactor candidate. This prey attracted our interest for further investigation because KLF2 is a nuclear transcriptional factor whereas most known Smurf1 substrates including Smad1/5, MEKK2, Prickle 1, TRAF4, and RhoA are localized in the

Discussion

In this study, we identified the HECT-type ubiquitin ligase Smurf1 could target the Krüppel-like factor KLF2 for ubiquitination and degradation in a proteasome-dependent manner. Notably, this regulation seems to be cell type-dependent since it was observed in human lung cancer H1299 cells but not human embryonic kidney HEK293T cells. The possible cause might be the different distribution pattern of Smurf1 in these cells (Fig. 3). Depletion of Smurf1 in H1299 resulted in a significant

Acknowledgments

This research was supported by the Chinese National Basic Research Programs (2011CB910602, 2007CB914601, 2010CB912202), the Chinese National Natural Science Foundation Projects (30830029, 30970601, 31070693) and the National Key Technologies R&D Program for New Drugs (2009ZX09503-002, 2009ZX09301-002).

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