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Phosphorylation of the Drosophila JNKKK Slipper is essential for proper morphogenesis and heat shock response

Gonda, Rebecca L (2011) Phosphorylation of the Drosophila JNKKK Slipper is essential for proper morphogenesis and heat shock response. Doctoral Dissertation, University of Pittsburgh.

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    Abstract

    Signal transduction pathways rely on proper protein activation and regulation to elicit appropriate responses to extracellular cues. Kinases within these pathways are commonly activated through a multi-step mechanism, which involves phosphorylation. Additionally, regulatory modifications can occur to modulate signals, ensuring that the proper intensity and duration of signaling is achieved in the correct context. I have identified two modes of regulation of the Drosophila JNKKK, Slipper. First, investigation into kinase domain phosphorylation reveals that conserved putative phosphorylation sites are required for Slpr function. Site-directed mutagenesis converting three serine/threonine residues to alanines (SlprAAA) renders the protein inactive, and SlprAAA behaves as a dominant negative during several Slpr-mediated processes. Transgenic flies expressing SlprAAA display phenotypes associated with a loss of JNK signaling such as dorsal open embryos that lose JNK pathway marker gene expression, cleft thorax indicative of a loss of JNK signaling during thorax closure, and a failure to rescue slpr mutants. Importantly, double alanine mutant analysis (TST mutated to AAT, ASA, and TAA) reveals that T295 is crucial for Slpr signaling as the two double mutants that contain alanine mutations at that residue are nonfunctional while SlprAAT retains some Slpr function.An additional phosphorylation site outside of the kinase domain was identified at a conserved MAPK consensus site, PXSP. Though flies expressing a non-phosphorylatable PXAP develop normally and have no defects in Slpr-dependent functions in morphogenesis, both embryos and adults are sensitive to heat shock. Conversely, a phospho-mimetic version, PXEP, confers thermoresistance. Biochemical assays implicate the Jun kinase, Bsk, as the MAPK required for this signaling to PXSP. These results suggest that Bsk phosphorylates Slpr in a positive feedback loop during heat stress to maintain homeostasis. Together, these experiments demonstrate the need for Slpr phosphorylation in two circumstances. Not only is phosphorylation required to maintain Slpr-dependent JNK signaling in morphological processes through proper protein activation, but it is also essential in a context-specific manner for a previously unidentified role of Slpr and the Drosophila JNK pathway in heat shock response.


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    Item Type: University of Pittsburgh ETD
    Creators/Authors:
    CreatorsEmailORCID
    Gonda, Rebecca Lreg11@pitt.edu
    ETD Committee:
    ETD Committee TypeCommittee MemberEmailORCID
    Committee ChairStronach, Bethstronach@pitt.edu
    Committee MemberChapman, Deborahdlc7@pitt.edu
    Committee MemberHildebrand, Jeffreyjeffh@pitt.edu
    Committee MemberMinden, Jonminden@cmu.edu
    Committee MemberGrabowski, Paulapag4@pitt.edu
    Title: Phosphorylation of the Drosophila JNKKK Slipper is essential for proper morphogenesis and heat shock response
    Status: Unpublished
    Abstract: Signal transduction pathways rely on proper protein activation and regulation to elicit appropriate responses to extracellular cues. Kinases within these pathways are commonly activated through a multi-step mechanism, which involves phosphorylation. Additionally, regulatory modifications can occur to modulate signals, ensuring that the proper intensity and duration of signaling is achieved in the correct context. I have identified two modes of regulation of the Drosophila JNKKK, Slipper. First, investigation into kinase domain phosphorylation reveals that conserved putative phosphorylation sites are required for Slpr function. Site-directed mutagenesis converting three serine/threonine residues to alanines (SlprAAA) renders the protein inactive, and SlprAAA behaves as a dominant negative during several Slpr-mediated processes. Transgenic flies expressing SlprAAA display phenotypes associated with a loss of JNK signaling such as dorsal open embryos that lose JNK pathway marker gene expression, cleft thorax indicative of a loss of JNK signaling during thorax closure, and a failure to rescue slpr mutants. Importantly, double alanine mutant analysis (TST mutated to AAT, ASA, and TAA) reveals that T295 is crucial for Slpr signaling as the two double mutants that contain alanine mutations at that residue are nonfunctional while SlprAAT retains some Slpr function.An additional phosphorylation site outside of the kinase domain was identified at a conserved MAPK consensus site, PXSP. Though flies expressing a non-phosphorylatable PXAP develop normally and have no defects in Slpr-dependent functions in morphogenesis, both embryos and adults are sensitive to heat shock. Conversely, a phospho-mimetic version, PXEP, confers thermoresistance. Biochemical assays implicate the Jun kinase, Bsk, as the MAPK required for this signaling to PXSP. These results suggest that Bsk phosphorylates Slpr in a positive feedback loop during heat stress to maintain homeostasis. Together, these experiments demonstrate the need for Slpr phosphorylation in two circumstances. Not only is phosphorylation required to maintain Slpr-dependent JNK signaling in morphological processes through proper protein activation, but it is also essential in a context-specific manner for a previously unidentified role of Slpr and the Drosophila JNK pathway in heat shock response.
    Date: 29 June 2011
    Date Type: Completion
    Defense Date: 01 February 2011
    Approval Date: 29 June 2011
    Submission Date: 18 April 2011
    Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
    Patent pending: No
    Institution: University of Pittsburgh
    Thesis Type: Doctoral Dissertation
    Refereed: Yes
    Degree: PhD - Doctor of Philosophy
    URN: etd-04182011-172258
    Uncontrolled Keywords: MAPK signaling; thermostress
    Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
    Date Deposited: 10 Nov 2011 14:38
    Last Modified: 22 May 2012 16:10
    Other ID: http://etd.library.pitt.edu/ETD/available/etd-04182011-172258/, etd-04182011-172258

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