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Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-56121

Schlaefli, P. The role of PASKIN in metabolism and protein synthesis. 2011, University of Zurich, Faculty of Science.

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Abstract

PER-ARNT-SIM (PAS) domains are occurring throughout all kingdoms of life. Bacterial and archaeal PAS domains mainly act as sensory modules of a variety of environmental stimuli
either directly by binding a ligand to be sensed or indirectly involving co-factors. Sensing induces conformational changes of the PAS domain that are transduced to effector domains, which are often kinases. In other cases, especially in eukaryotic basic helix-loop-helix
transcription factors, PAS domains can act as dimerisation interfaces. PASKIN is conserved from yeast to man and displays the unique combination for eukaryotic proteins of a Per-Arnt-Sim (PAS) domain and a Ser/Thr kinase function. For the PAS domain of PASKIN, a model proposed sensing and binding of a yet unknown metabolic ligand that might activate the kinase domain. For the kinase function, a number of proteins involved in glycogen metabolism and protein synthesis were identified as phosphorylation targets in yeast. For mammalian PASKIN, two targets involved in metabolism have been identified. The first
target, similar to yeast, is mammalian glycogen synthase, and the second target is pancreatic and duodenal homeobox protein 1 (PDX-1), an important transcription factor for insulin expression. Accordingly, it has been reported that PASKIN levels are induced in pancreatic β-cells upon glucose stimulation, itself induces insulin expression and secondarily affects insulin secretion. Recent findings also reported an inhibitory function of PASKIN in glucagon
secretion, suggesting that PASKIN might regulate energy homeostasis not only in yeast, but in mammals as well. Furthermore, whole body homeostasis has been investigated making use of our Paskin-/- mouse model. It has been shown that male Paskin-/- mice when fed a high fat diet (HFD) were protected from detrimental effects of the metabolic syndrome. Paskin-/- male mice displayed a lower body weight and a better glucose and insulin tolerance than wildtype
littermates and showed a hypermetabolic phenotype in indirect calorimetry experiments.
Using a similar approach, we herein aimed to reproduce the hypermetabolic phenotype on HFD feeding. Whereas we could see a lower gain of body weight for Paskin-/- animals and a
better glucose tolerance after 45% fat by calories HFD feeding, we could not increase the effects upon feeding a 60% HFD and failed to see any difference in indirect calorimetry or body temperature. We therefore speculate that PASKIN might play a more subtle role in
energy homeostasis that might be affected by housing, diet or age of the animals. We therefore focused on the molecular and cellular level aiming to answer the following
questions: 1) What is the substrate specificity and what might be potential targets of metabolism and protein translation for the PASKIN kinase function? 2) What could be the potentially activating ligand for the PAS domain of PASKIN? Herein, we show that the human eukaryotic translation elongation factor 1A1 is a novel interaction partner and kinase target of PASKIN and is able to increase total translation in vitro. To screen for other putative
PASKIN phosphorylation targets, we assessed the kinase function on a target peptide microarray. We found a substrate specificity similar to the consensus sequences of protein kinase A (PKA) and C (PKC) and identified new metabolic targets. We further identified phosphorylation of ribosomal protein S6 at serines 235/236. Phosphorylation of S6 usually originates from p70 S6 kinases and is a marker for mTOR activity, and therefore often occurring in proliferating cells. However, comparing Paskin-/- versus Paskin+/+ MEFs we could not observe any differences in total translation, cell size or cell growth. Since PKC is
known to phosphorylate eEF1A1 at the same site as PASKIN and shows a similar substrate preference, we wondered wether PASKIN also requires similar phospholipid co-activators as PKC. Indeed, we found that PASKIN binds phosphatidylinositol monophosphates, but not as
we assumed to the PAS domain but to the kinase domain. We also investigated the effects of phosphatidylinositol phosphates both on auto- as well as on target phosphorylation in vitro.
Interestingly, while auto-phosphorylation is induced by the presence of phosphatidylinositol monophosphates and reduced by di- and tri-phosphorylated phosphatidylinositides, target
phosphorylation is reduced by the presence of all phosphatidylinositides tested. Since we found that PASKIN mainly localises along the cytoskeleton, either at the cell membrane or along stess fibers, this suggests that PASKIN might be involved in vesicular transport or cytoskeletal dynamics and its activity might be regulated accordingly dependent on phospholipid binding.
Conclusively, the identification of eEF1A1 and S6 as novel mammalian phosphorylation targets confirmed a role for PASKIN in the regulation of protein translation. The observation that PASKIN can bind phosphatidylinositides opens novel perspectives for future research as
well, and might be connected to a metabolic phenotype by interfering with glucagon granule secretion or GLUT4 exocytosis. Further experiments to find conditions upregulating PASKIN and the identification of an activating ligand would certainly help to understand the general
mechanistics of PASKIN as a sensor of energy homeostasis.

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Additional indexing

Item Type:Dissertation
Referees:Wenger R H, Wagner C A, Lang F, Stiehl D P
Communities & Collections:04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology

04 Faculty of Medicine > Center for Integrative Human Physiology
DDC:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2011
Deposited On:22 Jan 2012 18:50
Last Modified:17 Oct 2012 15:40
Number of Pages:147
Related URLs:http://opac.nebis.ch/F/?local_base=NEBIS&CON_LNG=GER&func=find-b&find_code=SYS&request=006529615

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