Abstract
BackgroundIkappaB 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.ResultsWe 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.ConclusionWe 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.
Highlights
IkappaB kinases (IKKs) regulate the activity of Rel/NF-kappaB transcription factors by targeting their inhibitory partner proteins, IkappaBs, for degradation
Our results demonstrated that it is possible to co-precipitate Ik2 together with SpnF (Fig 1A), indicating that Spn-F interacts with Ik2, in agreement with the findings of the yeast two-hybrid assay
SFpignu-Freint1eracts physically with Ik2 and the C-terminus of Spn-F is crucial for this interaction Spn-F interacts physically with Ik2 and the C-terminus of Spn-F is crucial for this interaction. (A) Co-immunoprecipitation of green fluorescent protein (GFP)-Ik2 with HA-Spn-F
Summary
IkappaB kinases (IKKs) regulate the activity of Rel/NF-kappaB transcription factors by targeting their inhibitory partner proteins, IkappaBs, for degradation. Protein kinases of the IκB kinase (IKK) family are known for their roles in innate immune response signaling pathways in both mammals and Drosophila [1,2,3]. All mammalian IKKs studied so far have roles in immune responses, but operate on different targets. IKKα and IKKβ were identified in a protein complex that phosphorylates IκB and targets it for degradation, thereby allowing the nuclear localization and activation of NF-κB transcription factors [4,5,6]. Two members of the IKK family are known in Drosophila, namely DmIKKβ and Ik2 [10]. Ik ( known as DmIKKε) was shown to control actin and microtubule (MT) organization in an NF-κB-independent pathway [10,13,14]
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