Abstract
Leucine zipper-like transcriptional regulator 1 (LZTR1) encodes a member of the BTB-Kelch superfamily, which interacts with the Cullin3 (CUL3)-based E3 ubiquitin ligase complex. Mutations in LZTR1 have been identified in glioblastoma, schwannomatosis, and Noonan syndrome. However, the functional role of LZTR1 in carcinogenesis or human development is not fully understood. Here, we demonstrate that LZTR1 facilitates the polyubiquitination and degradation of RAS via the ubiquitin-proteasome pathway, leading to the inhibition of the RAS/MAPK signaling. The polyubiquitination and degradation of RAS was also observed in cells expressing MRAS, HRAS, NRAS, and KRAS as well as oncogenic RAS mutants and inhibited the activation of ERK1/2 and cell growth. In vivo ubiquitination assays showed that MRAS-K127 and HRAS-K170 were ubiquitinated by LZTR1 and that the polyubiquitinated-chains contained mainly Ub-K48, K63, and K33-linked chains, suggesting its possible involvement in autophagy. Immunoprecipitation analyses showed the interaction of LZTR1 and RAS-GTPases with autophagy-related proteins, including LC3B and SQSTM1/p62. Co-expression of LZTR1 and RAS increased the expression of lipidated form of LC3B. However, long-term treatment with chloroquine had little effect on RAS protein levels, suggesting that the contribution of autophagy to LZTR1-mediated RAS degradation is minimal. Taken together, these results show that LZTR1 functions as a “RAS killer protein” mainly via the ubiquitin-proteasome pathway regardless of the type of RAS GTPase, controlling downstream signal transduction. Our results also suggest a possible association of LZTR1 and RAS-GTPases with the autophagy. These findings provide clues for the elucidation of the mechanisms of RAS degradation and regulation of the RAS/MAPK signaling cascade.
Highlights
LZTR1 encodes leucine zipper-like transcription regulator 1 (LZTR1), which is a Golgi protein and that belongs to the Edited by V
We searched for molecules that were changed by LZTR1-siRNA and found that the protein levels of pan-RAS (HRAS, NRAS, and KRAS) were increased (Fig. 1a left panel)
We demonstrated that LZTR1 regulates the degradation of endogenous RAS and various types of expressed RAS proteins, including MRAS, HRAS, NRAS, and KRAS, mainly via the ubiquitin-proteasome pathway
Summary
LZTR1 encodes leucine zipper-like transcription regulator 1 (LZTR1), which is a Golgi protein and that belongs to the Edited by V. We have reported that LZTR1 interacts with the RAF1/SHOC2/PPP1CB complex and promotes RAF1Ser259 phosphorylation, leading to the inactivation of the MAPK signaling pathway [18]. Another observation was reported by two research groups, who found that LZTR1 promotes the site-specific mono- and di-ubiquitination of RAS and attenuates the association of ubiquitinated-RAS with the plasma membrane [19, 20]. It is not fully understood whether LZTR1 functionally interacts with RAS family proteins. We examined whether LZTR1 functionally interacts with RAS proteins and controls the activities of downstream signals, including the MAPK signaling pathway
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