Tuberous Sclerosis Tumor Suppressor Complex-like Complexes Act as GTPase-activating Proteins for Ral GTPases

Ryutaro Shirakawa,Shuya Fukai,Hirokazu Kondo,Ei Nakayama,Katsuya Okawa,Osamu Nureki,Takeshi Kimura,Tomohito Higashi,Tomoyuki Ikeda,Hisanori Horiuchi,Mitsunori Kawato,Toru Kita

doi:10.1074/jbc.m109.012112

Ryutaro Shirakawa, Shuya Fukai + Show 10 more

Open Access

https://doi.org/10.1074/jbc.m109.012112

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Abstract

The small GTPases RalA and RalB are multifunctional proteins regulating a variety of cellular processes. Like other GTPases, the activity of Ral is regulated by the opposing effects of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Although several RalGEFs have been identified and characterized, the molecular identity of RalGAP remains unknown. Here, we report the first molecular identification of RalGAPs, which we have named RalGAP1 and RalGAP2. They are large heterodimeric complexes, each consisting of a catalytic alpha1 or alpha2 subunit and a common beta subunit. These RalGAP complexes share structural and catalytic similarities with the tuberous sclerosis tumor suppressor complex, which acts as a GAP for Rheb. In vitro GTPase assays revealed that recombinant RalGAP1 accelerates the GTP hydrolysis rate of RalA by 280,000-fold. Heterodimerization was required for this GAP activity. In PC12 cells, knockdown of the beta subunit led to sustained Ral activation upon epidermal growth factor stimulation, indicating that the RalGAPs identified here are critical for efficient termination of Ral activation induced by extracellular stimuli. Our identification of RalGAPs will enable further understanding of Ral signaling in many biological and pathological processes.

Highlights

Members of the Ras superfamily of small GTPases act as molecular switches by cycling between an inactive GDP-bound
The GDP/ GTP cycling is strictly regulated by two classes of regulatory proteins termed guanine nucleotide exchange factors (GEFs)7 and GTPase-activating proteins (GAPs) [1]
The finding that Ras can activate most RalGEFs in addition to Raf and phosphoinositide 3-kinases suggested that Ral GTPases mediate some functions of oncogenic Ras

Summary

Introduction

Members of the Ras superfamily of small GTPases act as molecular switches by cycling between an inactive GDP-bound. Identification of RalGAPs existence of Ral-specific GAP activity in the cytosolic fraction of brain and testis. Cells were harvested in buffer C at the indicated time points, and the GTP-bound forms of RalA and Ras were pulled down using glutathione beads precoated with both GST-Sec5 Ral binding domain and GST-Raf1 Ras binding domain (20 ␮g each).

Results

Conclusion