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The Drosophila IKK-related kinase (Ik2) and Spindle-F proteins are part of a complex that regulates cytoskeleton organization during oogenesis.

Dubin-Bar D, Bitan A, Bakhrat A, Kaiden-Hasson R, Etzion S, Shaanan B, Abdu U - BMC Cell Biol. (2008)

Bottom Line: We showed that Ik2 phosphorylates Spn-F and demonstrated that this phosphorylation does not lead to Spn-F degradation.We conclude that Ik2 and Spn-F form a complex, which regulates cytoskeleton organization during Drosophila oogenesis and in which Spn-F is the direct regulatory target for Ik2.Interestingly, Ik2 in this complex does not function as a typical IKK in that it does not direct SpnF for degradation following phosphorylation.

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Affiliation: Department of Life Science, National Institute for Biotechnology in Negev, Ben-Gurion University, Beer-Sheva, 84105 Israel. bardik@bgu.ac.il

ABSTRACT

Background: IkappaB kinases (IKKs) regulate the activity of Rel/NF-kappaB transcription factors by targeting their inhibitory partner proteins, IkappaBs, for degradation. The Drosophila genome encodes two members of the IKK family. Whereas the first is a kinase essential for activation of the NF-kappaB pathway, the latter does not act as IkappaB kinase. Instead, recent findings indicate that Ik2 regulates F-actin assembly by mediating the function of nonapoptotic caspases via degradation of DIAP1. Also, it has been suggested that ik2 regulates interactions between the minus ends of the microtubules and the actin-rich cortex in the oocyte. Since spn-F mutants display oocyte defects similar to those of ik2 mutant, we decided to investigate whether Spn-F could be a direct regulatory target of Ik2.

Results: We found that Ik2 binds physically to Spn-F, biomolecular interaction analysis of Spn-F and Ik2 demonstrating that both proteins bind directly and form a complex. We showed that Ik2 phosphorylates Spn-F and demonstrated that this phosphorylation does not lead to Spn-F degradation. Ik2 is localized to the anterior ring of the oocyte and to punctate structures in the nurse cells together with Spn-F protein, and both proteins are mutually required for their localization.

Conclusion: We conclude that Ik2 and Spn-F form a complex, which regulates cytoskeleton organization during Drosophila oogenesis and in which Spn-F is the direct regulatory target for Ik2. Interestingly, Ik2 in this complex does not function as a typical IKK in that it does not direct SpnF for degradation following phosphorylation.

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Ik2 protein is co-localized with spn-F in the nurse cells and at the anterior ring of the oocyte. (A) Live imaging of stage 9 egg chamber from nanos-Gal4; pUASP GFP-Ik2 transgenic flies. Ik2 is localized to the anterior end of the oocyte. (B-D) Confocal image of stage 8 egg chamber from nanos-Gal4; pUASP GFP-Ik2 transgenic flies stained with anti-Spn-F. (B) GFP-Ik2 in green; (C) immunostaining for Spn-F in red; (D) merged picture. Both proteins are co-localized along the anterior cortex of the oocyte and in a punctate pattern in the nurse cells. Egg chambers are positioned that the anterior (A) is to the left left and posterior end (P) is to the right.
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Figure 6: Ik2 protein is co-localized with spn-F in the nurse cells and at the anterior ring of the oocyte. (A) Live imaging of stage 9 egg chamber from nanos-Gal4; pUASP GFP-Ik2 transgenic flies. Ik2 is localized to the anterior end of the oocyte. (B-D) Confocal image of stage 8 egg chamber from nanos-Gal4; pUASP GFP-Ik2 transgenic flies stained with anti-Spn-F. (B) GFP-Ik2 in green; (C) immunostaining for Spn-F in red; (D) merged picture. Both proteins are co-localized along the anterior cortex of the oocyte and in a punctate pattern in the nurse cells. Egg chambers are positioned that the anterior (A) is to the left left and posterior end (P) is to the right.

Mentions: In order to examine the localization of Ik2, transgenic flies expressing GFP-tagged Ik2 were generated. Expression of a GFP-tagged UASp-ik2 transgene under the direction of the ubiquitous actin-Gal4 driver rescued both the viability and bristle abnormalities of ik2 allelic combinations completely (data not shown). It has been reported that over-expression of Ik2 promotes DIAP1 elimination and induces cell death in somatic tissues [13]. However, we found that flies over-expressing GFP-tagged Ik2 or UAS-ik2 [10] in the germline are fertile and display no induction of apoptosis. We found that GFP-tagged Ik2 is localized to the anterior end of the oocyte and is present in a punctate pattern in the nurse cells, similarly to the pattern found for Spn-F (Fig. 6A). Next, we decided to test whether the GFP-tagged Ik2 co-localizes with endogenous Spn-F protein. For that purpose, ovaries expressing GFP-tagged Ik2 were immunostained with Spn-F antibody. We observed that Spn-F and Ik2 were co-localized at the anterior ring of the oocyte and in the same punctate structures in the nurse cells (Fig. 6B–D).


The Drosophila IKK-related kinase (Ik2) and Spindle-F proteins are part of a complex that regulates cytoskeleton organization during oogenesis.

Dubin-Bar D, Bitan A, Bakhrat A, Kaiden-Hasson R, Etzion S, Shaanan B, Abdu U - BMC Cell Biol. (2008)

Ik2 protein is co-localized with spn-F in the nurse cells and at the anterior ring of the oocyte. (A) Live imaging of stage 9 egg chamber from nanos-Gal4; pUASP GFP-Ik2 transgenic flies. Ik2 is localized to the anterior end of the oocyte. (B-D) Confocal image of stage 8 egg chamber from nanos-Gal4; pUASP GFP-Ik2 transgenic flies stained with anti-Spn-F. (B) GFP-Ik2 in green; (C) immunostaining for Spn-F in red; (D) merged picture. Both proteins are co-localized along the anterior cortex of the oocyte and in a punctate pattern in the nurse cells. Egg chambers are positioned that the anterior (A) is to the left left and posterior end (P) is to the right.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 6: Ik2 protein is co-localized with spn-F in the nurse cells and at the anterior ring of the oocyte. (A) Live imaging of stage 9 egg chamber from nanos-Gal4; pUASP GFP-Ik2 transgenic flies. Ik2 is localized to the anterior end of the oocyte. (B-D) Confocal image of stage 8 egg chamber from nanos-Gal4; pUASP GFP-Ik2 transgenic flies stained with anti-Spn-F. (B) GFP-Ik2 in green; (C) immunostaining for Spn-F in red; (D) merged picture. Both proteins are co-localized along the anterior cortex of the oocyte and in a punctate pattern in the nurse cells. Egg chambers are positioned that the anterior (A) is to the left left and posterior end (P) is to the right.
Mentions: In order to examine the localization of Ik2, transgenic flies expressing GFP-tagged Ik2 were generated. Expression of a GFP-tagged UASp-ik2 transgene under the direction of the ubiquitous actin-Gal4 driver rescued both the viability and bristle abnormalities of ik2 allelic combinations completely (data not shown). It has been reported that over-expression of Ik2 promotes DIAP1 elimination and induces cell death in somatic tissues [13]. However, we found that flies over-expressing GFP-tagged Ik2 or UAS-ik2 [10] in the germline are fertile and display no induction of apoptosis. We found that GFP-tagged Ik2 is localized to the anterior end of the oocyte and is present in a punctate pattern in the nurse cells, similarly to the pattern found for Spn-F (Fig. 6A). Next, we decided to test whether the GFP-tagged Ik2 co-localizes with endogenous Spn-F protein. For that purpose, ovaries expressing GFP-tagged Ik2 were immunostained with Spn-F antibody. We observed that Spn-F and Ik2 were co-localized at the anterior ring of the oocyte and in the same punctate structures in the nurse cells (Fig. 6B–D).

Bottom Line: We showed that Ik2 phosphorylates Spn-F and demonstrated that this phosphorylation does not lead to Spn-F degradation.We conclude that Ik2 and Spn-F form a complex, which regulates cytoskeleton organization during Drosophila oogenesis and in which Spn-F is the direct regulatory target for Ik2.Interestingly, Ik2 in this complex does not function as a typical IKK in that it does not direct SpnF for degradation following phosphorylation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Science, National Institute for Biotechnology in Negev, Ben-Gurion University, Beer-Sheva, 84105 Israel. bardik@bgu.ac.il

ABSTRACT

Background: IkappaB kinases (IKKs) regulate the activity of Rel/NF-kappaB transcription factors by targeting their inhibitory partner proteins, IkappaBs, for degradation. The Drosophila genome encodes two members of the IKK family. Whereas the first is a kinase essential for activation of the NF-kappaB pathway, the latter does not act as IkappaB kinase. Instead, recent findings indicate that Ik2 regulates F-actin assembly by mediating the function of nonapoptotic caspases via degradation of DIAP1. Also, it has been suggested that ik2 regulates interactions between the minus ends of the microtubules and the actin-rich cortex in the oocyte. Since spn-F mutants display oocyte defects similar to those of ik2 mutant, we decided to investigate whether Spn-F could be a direct regulatory target of Ik2.

Results: We found that Ik2 binds physically to Spn-F, biomolecular interaction analysis of Spn-F and Ik2 demonstrating that both proteins bind directly and form a complex. We showed that Ik2 phosphorylates Spn-F and demonstrated that this phosphorylation does not lead to Spn-F degradation. Ik2 is localized to the anterior ring of the oocyte and to punctate structures in the nurse cells together with Spn-F protein, and both proteins are mutually required for their localization.

Conclusion: We conclude that Ik2 and Spn-F form a complex, which regulates cytoskeleton organization during Drosophila oogenesis and in which Spn-F is the direct regulatory target for Ik2. Interestingly, Ik2 in this complex does not function as a typical IKK in that it does not direct SpnF for degradation following phosphorylation.

Show MeSH