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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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STRADα and STRADβ are redundant in axogenesis and limiting programmed cell death. Embryonic day 18.5 coronal sections immunolabeled for either corticofugal fibers (Tag1, green) (A-D, A’-D’) or activated caspase3 (Act-Casp3, green) (E-H, E’-H’) and a nuclear marker (DRAQ5, magenta in A-D, blue in E-H). Axons are normal in wild-type, STRADα KO and STRADβ cKO brains, but completely gone in the STRADα/STRADβ double KO brain (A-D). Scale bar = 100 μm. Activated caspase3 is largely absent from wild-type, STRADα KO and STRADβ cKO brain, but drastically increased in STRADα/β double KO cortex (E-H, E’-H’). Scale bar = 100 μm. Higher magnifications of cortical regions of TAG1 immunolabeling and activated caspase 3 are shown in grayscale to the right (A’-D’ and E’-H’).
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Figure 3: STRADα and STRADβ are redundant in axogenesis and limiting programmed cell death. Embryonic day 18.5 coronal sections immunolabeled for either corticofugal fibers (Tag1, green) (A-D, A’-D’) or activated caspase3 (Act-Casp3, green) (E-H, E’-H’) and a nuclear marker (DRAQ5, magenta in A-D, blue in E-H). Axons are normal in wild-type, STRADα KO and STRADβ cKO brains, but completely gone in the STRADα/STRADβ double KO brain (A-D). Scale bar = 100 μm. Activated caspase3 is largely absent from wild-type, STRADα KO and STRADβ cKO brain, but drastically increased in STRADα/β double KO cortex (E-H, E’-H’). Scale bar = 100 μm. Higher magnifications of cortical regions of TAG1 immunolabeling and activated caspase 3 are shown in grayscale to the right (A’-D’ and E’-H’).

Mentions: To address the in vivo complementation of the STRAD paralogs, we generated two novel mouse lines (a STRADα- line and a conditional allele of STRADβ (see Additional file 1: Figure S3A)). The STRADα constitutive animals do not produce STRADα protein (see Additional file 1: Figure S3B) and expire perinatally (see Additional file 1: Figure S3C), while the STRADβ mice have no overt phenotypes. Histologic characterization of cerebral cortices indicated that elimination of either STRADα (Figure 3B,B’) or STRADβ (Figure 3C,C’) alone is not sufficient to disrupt axogenesis. However, deletion of both STRAD genes caused a profound loss of TAG1-positive projection axons (Figure 3D,D’), mirroring the effect observed following conditional deletion of LKB1 [5]. Similarly, neuronal polarization defects (indeterminate neurites) were observed in primary cortical cultures of STRADα/β double KO cortices compared to controls when immuno-stained for the axon/dendrite markers Tau1 and microtubule associated protein 2 (MAP2), respectively (see Additional file 1: Figure S3E-G). This indicates that either STRADα or β is sufficient to drive axogenesis during corticogenesis.


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 and STRADβ are redundant in axogenesis and limiting programmed cell death. Embryonic day 18.5 coronal sections immunolabeled for either corticofugal fibers (Tag1, green) (A-D, A’-D’) or activated caspase3 (Act-Casp3, green) (E-H, E’-H’) and a nuclear marker (DRAQ5, magenta in A-D, blue in E-H). Axons are normal in wild-type, STRADα KO and STRADβ cKO brains, but completely gone in the STRADα/STRADβ double KO brain (A-D). Scale bar = 100 μm. Activated caspase3 is largely absent from wild-type, STRADα KO and STRADβ cKO brain, but drastically increased in STRADα/β double KO cortex (E-H, E’-H’). Scale bar = 100 μm. Higher magnifications of cortical regions of TAG1 immunolabeling and activated caspase 3 are shown in grayscale to the right (A’-D’ and E’-H’).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: STRADα and STRADβ are redundant in axogenesis and limiting programmed cell death. Embryonic day 18.5 coronal sections immunolabeled for either corticofugal fibers (Tag1, green) (A-D, A’-D’) or activated caspase3 (Act-Casp3, green) (E-H, E’-H’) and a nuclear marker (DRAQ5, magenta in A-D, blue in E-H). Axons are normal in wild-type, STRADα KO and STRADβ cKO brains, but completely gone in the STRADα/STRADβ double KO brain (A-D). Scale bar = 100 μm. Activated caspase3 is largely absent from wild-type, STRADα KO and STRADβ cKO brain, but drastically increased in STRADα/β double KO cortex (E-H, E’-H’). Scale bar = 100 μm. Higher magnifications of cortical regions of TAG1 immunolabeling and activated caspase 3 are shown in grayscale to the right (A’-D’ and E’-H’).
Mentions: To address the in vivo complementation of the STRAD paralogs, we generated two novel mouse lines (a STRADα- line and a conditional allele of STRADβ (see Additional file 1: Figure S3A)). The STRADα constitutive animals do not produce STRADα protein (see Additional file 1: Figure S3B) and expire perinatally (see Additional file 1: Figure S3C), while the STRADβ mice have no overt phenotypes. Histologic characterization of cerebral cortices indicated that elimination of either STRADα (Figure 3B,B’) or STRADβ (Figure 3C,C’) alone is not sufficient to disrupt axogenesis. However, deletion of both STRAD genes caused a profound loss of TAG1-positive projection axons (Figure 3D,D’), mirroring the effect observed following conditional deletion of LKB1 [5]. Similarly, neuronal polarization defects (indeterminate neurites) were observed in primary cortical cultures of STRADα/β double KO cortices compared to controls when immuno-stained for the axon/dendrite markers Tau1 and microtubule associated protein 2 (MAP2), respectively (see Additional file 1: Figure S3E-G). This indicates that either STRADα or β is sufficient to drive axogenesis during corticogenesis.

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