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STRAD pseudokinases regulate axogenesis and LKB1 stability.

Veleva-Rotse BO, Smart JL, Baas AF, Edmonds B, Zhao ZM, Brown A, Klug LR, Hansen K, Reilly G, Gardner AP, Subbiah K, Gaucher EA, Clevers H, Barnes AP - Neural Dev (2014)

Bottom Line: We find that STRADα is highly spliced and appears to be the primal STRAD paralog.We also reveal a reciprocal protein-stabilizing relationship in vivo between LKB1 and STRADα, whereby STRADα specifically maintains LKB1 protein levels via cytoplasmic compartmentalization.We demonstrate a novel role for STRADβ in axogenesis and also show for the first time in vivo that STRADα, but not STRADβ, is responsible for LKB1 protein stability.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics-Doernbecher, Children's Hospital, Portland, OR 97239, USA. barnesan@ohsu.edu.

ABSTRACT

Background: Neuronal polarization is an essential step of morphogenesis and connectivity in the developing brain. The serine/threonine kinase LKB1 is a key regulator of cell polarity, metabolism, tumorigenesis, and is required for axon formation. It is allosterically regulated by two related and evolutionarily conserved pseudokinases, STe20-Related ADapters (STRADs) α and β. The roles of STRADα and STRADβ in the developing nervous system are not fully defined, nor is it known whether they serve distinct functions.

Results: We find that STRADα is highly spliced and appears to be the primal STRAD paralog. We report that each STRAD is sufficient for axogenesis and promoting cell survival in the developing cortex. We also reveal a reciprocal protein-stabilizing relationship in vivo between LKB1 and STRADα, whereby STRADα specifically maintains LKB1 protein levels via cytoplasmic compartmentalization.

Conclusions: We demonstrate a novel role for STRADβ in axogenesis and also show for the first time in vivo that STRADα, but not STRADβ, is responsible for LKB1 protein stability.

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Related in: MedlinePlus

STRADα, but not STRADβ stabilizes LKB1 protein. (A) Representative Western blot of lysates from wild-type, STRADα KO, STRADβ cKO, or STRADα/β double KO embryonic day 16.5 (E16.5) cortex. Actin is a loading control. (B) Quantification of LKB1 protein levels analyzed by Western blot, normalized to wild-type cortical lysate. N ≥15 cortices from at least three litters of each genotype. There is no significant difference between columns 1 and 3 or columns 2 and 4. ***P <0.0001 using one-way ANOVA with Bonferroni’s multiple comparison test. (C) Western blot of Nestin-cre+; STRADαf+ or STRADαff lysates across developmental time. Quantification of LKB1 protein, normalized to postnatal day 0 (P0) STRADαf+; Nestin-cre+. N ≥3 brains of each genotype from three litters for each time-point. (D) Western blot of E16.5 cortical lysates showing STRADα protein is significantly reduced following conditional cortical loss of LKB1. (E and F) Quantification of N ≥4 independent LKB1 stability time-courses in HEK cells transfected with epitope-tagged LKB1 protein following cycloheximide-mediated translation inhibition, with three to six replicates of each condition in each experiment. (E) STRADα-2 significantly increases LKB1 half-life, as does STRADβ-1, to a lesser extent. (F) LKB1ΔNLS is as stable as LKB1 + STRADα-2 and LKB1ΔNLS + STRADα-2 is similar to LKB1ΔNLS + Vector. Error bars represent SEM. Repeated measures ANOVA with Dunnett’s multiple comparison test with LKB1 + empty vector as the control was employed. ***P <0.001, **P <0.005. ANOVA, analysis of variance; KO, knockout; SEM, standard error of the mean.
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Figure 4: STRADα, but not STRADβ stabilizes LKB1 protein. (A) Representative Western blot of lysates from wild-type, STRADα KO, STRADβ cKO, or STRADα/β double KO embryonic day 16.5 (E16.5) cortex. Actin is a loading control. (B) Quantification of LKB1 protein levels analyzed by Western blot, normalized to wild-type cortical lysate. N ≥15 cortices from at least three litters of each genotype. There is no significant difference between columns 1 and 3 or columns 2 and 4. ***P <0.0001 using one-way ANOVA with Bonferroni’s multiple comparison test. (C) Western blot of Nestin-cre+; STRADαf+ or STRADαff lysates across developmental time. Quantification of LKB1 protein, normalized to postnatal day 0 (P0) STRADαf+; Nestin-cre+. N ≥3 brains of each genotype from three litters for each time-point. (D) Western blot of E16.5 cortical lysates showing STRADα protein is significantly reduced following conditional cortical loss of LKB1. (E and F) Quantification of N ≥4 independent LKB1 stability time-courses in HEK cells transfected with epitope-tagged LKB1 protein following cycloheximide-mediated translation inhibition, with three to six replicates of each condition in each experiment. (E) STRADα-2 significantly increases LKB1 half-life, as does STRADβ-1, to a lesser extent. (F) LKB1ΔNLS is as stable as LKB1 + STRADα-2 and LKB1ΔNLS + STRADα-2 is similar to LKB1ΔNLS + Vector. Error bars represent SEM. Repeated measures ANOVA with Dunnett’s multiple comparison test with LKB1 + empty vector as the control was employed. ***P <0.001, **P <0.005. ANOVA, analysis of variance; KO, knockout; SEM, standard error of the mean.

Mentions: Given the similarity with LKB1 phenotypes, we tested whether endogenous LKB1 expression is affected by loss of either or both STRAD proteins in vivo. We find loss of STRADα leads to a significant decrease (approximately 85%) in LKB1 protein levels in the embryonic (embryonic day (E16.5) cerebral cortex (Figure 4A-B), a result mirrored in other tissues (see Additional file 1: Figure S4B). This observation is despite a significant increase in LKB1 transcription (see Additional file 1: Figure S4A), indicating that regulation of protein expression occurs predominantly post-transcriptionally. Surprisingly, this reduction in LKB1 levels impairs neither axogenesis nor cell survival (Figure 3).


STRAD pseudokinases regulate axogenesis and LKB1 stability.

Veleva-Rotse BO, Smart JL, Baas AF, Edmonds B, Zhao ZM, Brown A, Klug LR, Hansen K, Reilly G, Gardner AP, Subbiah K, Gaucher EA, Clevers H, Barnes AP - Neural Dev (2014)

STRADα, but not STRADβ stabilizes LKB1 protein. (A) Representative Western blot of lysates from wild-type, STRADα KO, STRADβ cKO, or STRADα/β double KO embryonic day 16.5 (E16.5) cortex. Actin is a loading control. (B) Quantification of LKB1 protein levels analyzed by Western blot, normalized to wild-type cortical lysate. N ≥15 cortices from at least three litters of each genotype. There is no significant difference between columns 1 and 3 or columns 2 and 4. ***P <0.0001 using one-way ANOVA with Bonferroni’s multiple comparison test. (C) Western blot of Nestin-cre+; STRADαf+ or STRADαff lysates across developmental time. Quantification of LKB1 protein, normalized to postnatal day 0 (P0) STRADαf+; Nestin-cre+. N ≥3 brains of each genotype from three litters for each time-point. (D) Western blot of E16.5 cortical lysates showing STRADα protein is significantly reduced following conditional cortical loss of LKB1. (E and F) Quantification of N ≥4 independent LKB1 stability time-courses in HEK cells transfected with epitope-tagged LKB1 protein following cycloheximide-mediated translation inhibition, with three to six replicates of each condition in each experiment. (E) STRADα-2 significantly increases LKB1 half-life, as does STRADβ-1, to a lesser extent. (F) LKB1ΔNLS is as stable as LKB1 + STRADα-2 and LKB1ΔNLS + STRADα-2 is similar to LKB1ΔNLS + Vector. Error bars represent SEM. Repeated measures ANOVA with Dunnett’s multiple comparison test with LKB1 + empty vector as the control was employed. ***P <0.001, **P <0.005. ANOVA, analysis of variance; KO, knockout; SEM, standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4016016&req=5

Figure 4: STRADα, but not STRADβ stabilizes LKB1 protein. (A) Representative Western blot of lysates from wild-type, STRADα KO, STRADβ cKO, or STRADα/β double KO embryonic day 16.5 (E16.5) cortex. Actin is a loading control. (B) Quantification of LKB1 protein levels analyzed by Western blot, normalized to wild-type cortical lysate. N ≥15 cortices from at least three litters of each genotype. There is no significant difference between columns 1 and 3 or columns 2 and 4. ***P <0.0001 using one-way ANOVA with Bonferroni’s multiple comparison test. (C) Western blot of Nestin-cre+; STRADαf+ or STRADαff lysates across developmental time. Quantification of LKB1 protein, normalized to postnatal day 0 (P0) STRADαf+; Nestin-cre+. N ≥3 brains of each genotype from three litters for each time-point. (D) Western blot of E16.5 cortical lysates showing STRADα protein is significantly reduced following conditional cortical loss of LKB1. (E and F) Quantification of N ≥4 independent LKB1 stability time-courses in HEK cells transfected with epitope-tagged LKB1 protein following cycloheximide-mediated translation inhibition, with three to six replicates of each condition in each experiment. (E) STRADα-2 significantly increases LKB1 half-life, as does STRADβ-1, to a lesser extent. (F) LKB1ΔNLS is as stable as LKB1 + STRADα-2 and LKB1ΔNLS + STRADα-2 is similar to LKB1ΔNLS + Vector. Error bars represent SEM. Repeated measures ANOVA with Dunnett’s multiple comparison test with LKB1 + empty vector as the control was employed. ***P <0.001, **P <0.005. ANOVA, analysis of variance; KO, knockout; SEM, standard error of the mean.
Mentions: Given the similarity with LKB1 phenotypes, we tested whether endogenous LKB1 expression is affected by loss of either or both STRAD proteins in vivo. We find loss of STRADα leads to a significant decrease (approximately 85%) in LKB1 protein levels in the embryonic (embryonic day (E16.5) cerebral cortex (Figure 4A-B), a result mirrored in other tissues (see Additional file 1: Figure S4B). This observation is despite a significant increase in LKB1 transcription (see Additional file 1: Figure S4A), indicating that regulation of protein expression occurs predominantly post-transcriptionally. Surprisingly, this reduction in LKB1 levels impairs neither axogenesis nor cell survival (Figure 3).

Bottom Line: We find that STRADα is highly spliced and appears to be the primal STRAD paralog.We also reveal a reciprocal protein-stabilizing relationship in vivo between LKB1 and STRADα, whereby STRADα specifically maintains LKB1 protein levels via cytoplasmic compartmentalization.We demonstrate a novel role for STRADβ in axogenesis and also show for the first time in vivo that STRADα, but not STRADβ, is responsible for LKB1 protein stability.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics-Doernbecher, Children's Hospital, Portland, OR 97239, USA. barnesan@ohsu.edu.

ABSTRACT

Background: Neuronal polarization is an essential step of morphogenesis and connectivity in the developing brain. The serine/threonine kinase LKB1 is a key regulator of cell polarity, metabolism, tumorigenesis, and is required for axon formation. It is allosterically regulated by two related and evolutionarily conserved pseudokinases, STe20-Related ADapters (STRADs) α and β. The roles of STRADα and STRADβ in the developing nervous system are not fully defined, nor is it known whether they serve distinct functions.

Results: We find that STRADα is highly spliced and appears to be the primal STRAD paralog. We report that each STRAD is sufficient for axogenesis and promoting cell survival in the developing cortex. We also reveal a reciprocal protein-stabilizing relationship in vivo between LKB1 and STRADα, whereby STRADα specifically maintains LKB1 protein levels via cytoplasmic compartmentalization.

Conclusions: We demonstrate a novel role for STRADβ in axogenesis and also show for the first time in vivo that STRADα, but not STRADβ, is responsible for LKB1 protein stability.

Show MeSH
Related in: MedlinePlus