Gpn1 and Gpn3 associate tightly and their protein levels are mutually dependent in mammalian cells

Méndez-Hernández, Lucía E.; Pérez-Mejía, Ana E.; Lara‐Chacón, Bárbara; Barbosa-Camacho, Angel A.; Peña-Gómez, Sonia G.; Martínez-Sánchez, Mayra; Robledo-Rivera, Angélica Y.; Sánchez‐Olea, Roberto; Calera, Mónica R.

doi:10.1016/j.febslet.2014.08.038

Cited by 11 publications

(35 citation statements)

References 21 publications

(41 reference statements)

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“…Gpn3 was reported ubiquitinated on lysine 216, a finding confirmed in a second global study by Mertins et al [10]. Finally, we demonstrated that polyubiquitination of Gpn3 occurred in the cell nucleus and that it was prominent when expressing Gpn3 alone, but that this process was inhibited in a dose-dependent manner by co-transfecting Gpn1, a critical Gpn3 partner in human cells [7]. However, it is necessary to first confirm these results for individual proteins and then determine if this ubiquitination corresponds to mono, multi or polyubiquitination, as well as physiological relevance and regulation.…”

Section: Discussionsupporting

confidence: 87%

“…These results indicate that polyubiquitination on lysine 216 is a regulatory process for proteasomal degradation of Gpn3 that is not in a complex with Gpn1, a critical partner of Gpn3 in mammalian cells [7]. We showed that Gpn3 is polyubiquitinated in the presence of MG132.…”

mentioning

confidence: 82%

“…We previously reported that Gpn3 strongly interacts with Gpn1 to form a single protein complex in mammalian cells [7]. Additionally, suppression of Gpn1 expression led to a significant decrease in Gpn3 protein levels, demonstrating that Gpn3 stability does critically depend on the presence of Gpn1.…”

Section: Polyubiquitination Of Gpn3r-flag Is Prevented By Gpn1-eyfp Imentioning

confidence: 92%

“…For this purpose, we first generated retroviral molecular constructs to stably express Gpn3R-Flag or Gpn3R-Flag K216R in MCF-12A cells, as previously described [7]. For this purpose, we first generated retroviral molecular constructs to stably express Gpn3R-Flag or Gpn3R-Flag K216R in MCF-12A cells, as previously described [7].…”

Section: In Vivo Polyubiquitination Of Gpn3r-flag Occurs Mainly On Lymentioning

confidence: 99%

“…In mammalian cells, both Gpn1 and Gpn3 are required for the nuclear localization of RNAPII [4,6]. Gpn1 and Gpn3 strongly interact in human cells, and the stability of each protein depends on the presence of the other [7]. Gpn1 and Gpn3 strongly interact in human cells, and the stability of each protein depends on the presence of the other [7].…”

mentioning

confidence: 99%

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Gpn3 is polyubiquitinated on lysine 216 and degraded by the proteasome in the cell nucleus in a Gpn1‐inhibitable manner

et al. 2017

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Gpn1 associates with Gpn3, and both are required for RNA polymerase II nuclear targeting. Global studies have identified by mass spectrometry that human Gpn3 is ubiquitinated on lysines 189 and 216. Our goals here were to determine the type, physiological importance, and regulation of Gpn3 ubiquitination. After inhibiting the proteasome with MG132, Gpn3-Flag was polyubiquitinated on K216, but not K189, in HEK293T cells. Gpn3-Flag exhibited nucleo-cytoplasmic shuttling, but polyubiquitination and proteasomal degradation of Gpn3-Flag occurred only in the cell nucleus. Polyubiquitination-deficient Gpn3-Flag K216R displayed a longer half-life than Gpn3-Flag in two cell lines. Interestingly, Gpn1-EYFP inhibited Gpn3-Flag polyubiquitination in a dose-dependent manner. In conclusion, Gpn1-inhibitable, nuclear polyubiquitination on lysine 216 regulates the half-life of Gpn3 by tagging it for proteasomal degradation.

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Section: Discussionsupporting

confidence: 87%

mentioning

confidence: 82%

Section: Polyubiquitination Of Gpn3r-flag Is Prevented By Gpn1-eyfp Imentioning

confidence: 92%

Section: In Vivo Polyubiquitination Of Gpn3r-flag Occurs Mainly On Lymentioning

confidence: 99%

mentioning

confidence: 99%

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Gpn3 is polyubiquitinated on lysine 216 and degraded by the proteasome in the cell nucleus in a Gpn1‐inhibitable manner

et al. 2017

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The Gpn3 Q279* cancer‐associated mutant inhibits Gpn1 nuclear export and is deficient in RNA polymerase II nuclear targeting

et al. 2017

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Gpn3 is required for RNA polymerase II (RNAPII) nuclear targeting. Here, we investigated the effect of a cancer-associated Q279* nonsense mutation in Gpn3 cellular function. Employing RNAi, we replaced endogenous Gpn3 by wt or Q279* RNAi-resistant Gpn3R in epithelial model cells. RNAPII nuclear accumulation and transcriptional activity were markedly decreased in cells expressing only Gpn3R Q279*. Wild-type Gpn3R localized to the cytoplasm but a fraction of Gpn3R Q279* entered the cell nucleus and inhibited Gpn1-EYFP nuclear export. This property and the transcriptional deficit in Gpn3R Q279*-expressing cells required a PDZ-binding motif generated by the Q279* mutation. We conclude that an acquired PDZ-binding motif in Gpn3 Q279* caused Gpn3 nuclear entry, and inhibited Gpn1 nuclear export and Gpn3-mediated RNAPII nuclear targeting.

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Npa3/ScGpn1 carboxy-terminal tail is dispensable for cell viability and RNA polymerase II nuclear targeting but critical for microtubule stability and function

Guerrero-Serrano

Castanedo

Cristóbal‐Mondragón

et al. 2017

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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Genetic deletion of the essential GTPase Gpn1 or replacement of the endogenous gene by partial loss of function mutants in yeast is associated with multiple cellular phenotypes, including in all cases a marked cytoplasmic retention of RNA polymerase II (RNAPII). Global inhibition of RNAPII-mediated transcription due to malfunction of Gpn1 precludes the identification and study of other cellular function(s) for this GTPase. In contrast to the single Gpn protein present in Archaea, eukaryotic Gpn1 possesses an extension of approximately 100 amino acids at the C-terminal end of the GTPase domain. To determine the importance of this C-terminal extension in Saccharomyces cerevisiae Gpn1, we generated yeast strains expressing either C-terminal truncated (gpn1ΔC) or full-length ScGpn1. We found that ScGpn1ΔC was retained in the cell nucleus, an event physiologically relevant as gpn1ΔC cells contained a higher nuclear fraction of the RNAPII CTD phosphatase Rtr1. gpn1ΔC cells displayed an increased size, a delay in mitosis exit, and an increased sensitivity to the microtubule polymerization inhibitor benomyl at the cell proliferation level and two cellular events that depend on microtubule function: RNAPII nuclear targeting and vacuole integrity. These phenotypes were not caused by inhibition of RNAPII, as in gpn1ΔC cells RNAPII nuclear targeting and transcriptional activity were unaffected. These data, combined with our description here of a genetic interaction between GPN1 and BIK1, a microtubule plus-end tracking protein with a mitotic function, strongly suggest that the ScGpn1 C-terminal tail plays a critical role in microtubule dynamics and mitotic progression in an RNAPII-independent manner.

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Gpn1 and Gpn3 associate tightly and their protein levels are mutually dependent in mammalian cells

Cited by 11 publications

References 21 publications

Gpn3 is polyubiquitinated on lysine 216 and degraded by the proteasome in the cell nucleus in a Gpn1‐inhibitable manner

Gpn3 is polyubiquitinated on lysine 216 and degraded by the proteasome in the cell nucleus in a Gpn1‐inhibitable manner

The Gpn3 Q279* cancer‐associated mutant inhibits Gpn1 nuclear export and is deficient in RNA polymerase II nuclear targeting

Npa3/ScGpn1 carboxy-terminal tail is dispensable for cell viability and RNA polymerase II nuclear targeting but critical for microtubule stability and function

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