Regulation of membrane transporters by kinases

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URI: http://nbn-resolving.de/urn:nbn:de:bsz:21-opus-25544
http://hdl.handle.net/10900/48978
Dokumentart: Dissertation
Date: 2006
Language: English
Faculty: 7 Mathematisch-Naturwissenschaftliche Fakultät
Department: Sonstige - Biologie
Advisor: Lang, Florian
Day of Oral Examination: 2006-11-02
DDC Classifikation: 570 - Life sciences; biology
Keywords: Kinase
Other Keywords: Membrantransport , Creatin , Kreatin , Kreatinkinase
Creatine
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Inhaltszusammenfassung:

Creatin bindet Phosphate und dient so der Energiespeicherung. Die vorliegende Arbeit erforscht die Regulation von SLC6A8 durch das Serum und die glukose-abhängige Kinase SGK1.

Abstract:

Creatine binds phosphate thus serving energy storage. Cellular creatine uptake is ccomplished by the Na+,Cl-, creatine transporter CreaT (SLC6A8). The present study explored the regulation of SLC6A8 by the serum and glucocorticoid inducible kinase SGK1, a kinase upregulated during ischemia. In Xenopus oocytes expressing SLC6A8 but not in water injected oocytes creatine induced a current which was significantly enhanced by coexpression of wild type SGK1 and constitutively active S422DSGK1, but not inactive K127NSGK1. Kinetic analysis revealed that S422DSGK1 enhanced maximal current without significantly altering affinity. The effect of SGK1 was mimicked by the constitutively active isoform S419DSGK3 but not by inactive K119NSGK3, wild type isoform SGK2 or constitutively active related kinase T308D,S473DPKB. In conclusion, the kinases SGK1 and SGK3 increase SLC6A8 activity by increasing the maximal transport rate of the carrier. Deranged SGK1 and/or SGK3 dependent regulation of SLC6A8 may affect energy storage particularly in skeletal muscle, heart, and neurons. Cellular accumulation of creatine is accomplished by the Na+, Cl-, and creatine transporter CreaT (SLC6A8). The mammalian target of rapamycin (mTOR) is a kinase stimulating cellular nutrient uptake. The present experiments explored whether SLC6A8 is regulated by mTOR. In Xenopus oocytes expressing SLC6A8 but not in water injected oocytes, creatineinduced a current which was significantly enhanced by coexpression of mTOR. Kinetic analysis revealed that mTOR enhanced maximal current without significantly altering affinity. Preincubation of the oocytes for 32 h with rapamycin (50 nM) decreased the creatine-induced current and abrogated its stimulation by mTOR. The effect of mTOR on CreaT was blunted by additional coexpression of the inactive mutant of the serum and glucocorticoid inducible kinase K119NSGK1 and mimicked by coexpression of wild type SGK1. In conclusion, mTOR stimulates the creatine transporter SLC6A8 through mechanisms at least partially shared by the serum and glucocorticoid-inducible kinase SGK1. Adequate phosphate homeostasis is of critical importance for a wide variety of functions including bone mineralization and energy metabolism. Phosphate balance is a function of intestinal absorption and renal elimination, which are both under tight hormonal control. Intestinal phosphate absorption is accomplished by the Na+, phosphate cotransporter NaPi IIb (SLC34A2). Signaling mechanisms mediating hormonal regulation of SLC34A2 are incompletely understood. The mammalian target of rapamycin (mTOR) is a kinase regulating a variety of nutrient transporters. The present experiments explored whether mTOR regulates the activity of SLC34A2. In Xenopus oocytes expressing SLC34A2 but not in water injected oocytes phosphate (1 mM) induced a current (Ip) which was significantly enhanced by coexpression of mTOR. Preincubation of the oocytes for 24 h with rapamycin (50 nM) did not significantly affect Ip in the absence of mTOR but virtually abolished the increase of Ip following coexpression of mTOR. The wild type serum and glucocorticoid inducible kinase SGK1 and the constitutively active S422DSGK1 similarly stimulated Ip, an effect again reversed by rapamycin. Coexpression of the inactive mutant of the serum and glucocorticoid inducible kinase K119NSGK1 significantly decreased Ip and abrogated the stimulating effect of mTOR on Ip. In conclusion, mTOR and SGK1 cooperate in the stimulation of the intestinal phosphate transporter SLC34A2.

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