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Interacció de la proteïna quinasa CK2 amb la xaperona GRP94. Implicacions funcionals
| Content Provider | Semantic Scholar |
|---|---|
| Author | Armentia, Nerea Roher |
| Copyright Year | 2002 |
| Abstract | La proteina quinasa CK2 es una serina/treonina quinasa molt conservada i ubiqua en organismes eucariotes, implicada en diversos processos cel·lulars importants com proliferacio o tumorigenesis. Lenzim de mamifers esta format por dues subunitats catalitiques (a y/o a) y dues subunitats reguladores (b) encara que tambe sha descrit lexistencia de subunitats catalitiques lliures. La regulacio de lactivitat CK2 no es una regulacio classica mitjancant segons missatgers o fosforilacio, de fet la subunitat catalitica te activitat constitutiva. Es postula que la regulacio es podria dur a terme per variacio de la localitzacio subcel.lular mitjancant interaccio con proteines adscrites a compartiments subcel.lulares concrets. La proteina xaperona grp94 (94 kDa-glucose regulated protein) es una proteina destres que pertany a la familia de les hsp90 i que es localitza al Reticle Endoplasmic (RE) de totes les cel·lules eucariotes. Sexpressa constitutivament en condiciones normals y es sobreexpressa en situacions destres com deplecio de calci, inhibicio de la glicosilacio, infeccio virica, agents reductores etc. Utilitzant tecniques de Ressonancia Plasmonica, Far Western y Pull down demostrem que el domini carboxi terminal de grp94 interacciona amb la subunitat catalitica de CK2 (CK2a) pero no amb la subunitat reguladora ni amb lholoenzim (a2b2). Sha pogut mapejar la regio dinteraccio entre ambdues proteines, utilizant diversos mutants de CK2a, aixi la regio sha delimitat a una regio de lisines (K74-77) molt conservada entre las CK2a de diferents especies. Daltra banda sha demostrat que grp94 te activitat xaperona in vitro sobre CK2a i citrat sintasa (CS). Utilizant assajos dagregacio induida por xoc termic y tecniques de microscopia electronica sha determinat que grp94 es capac dinhibir lagregacion de CK2a i CS. Lactivitat xaperona depen fortament de lestat doligomeritzacio de grp94, de forma que en condicions reductores en que grp94 perd la seva estructura quaternaria, la capacitat dinhibir agregacio de proteines esta disminuida. Sha estudiat tambe la distribucio subcel·lular de CK2 i grp94 en un model animal de rates geneticament obeses (fa/fa). Aquestes rates son hiperinsulinemiques i resistents a insulina degut a una down-regulation del receptor de insulina. Shabia descrit en cel·lules en cultiu que el tractament amb dosis altes dinsulina induia la sinstesi de grp94. Sha observat que no hi ha variacio ni en els nivells ni en la distribucio subcel·lular de grp94 pero si hi ha variacio tan en els nivells com en la distribucio de CK2, que disminueix marcadament en citosol i augmenta en les fraccions membranoses. Protein kinase CK2 is a highly conserved and ubiquitously distributed serin/threonin kinase described in all eukaryotic organisms. CK2 has been implied in different cellular processes as proliferation or cancer. The mammalian enzyme is composed of two catalytic subunits (a and/or a) and two regulatory subunits (b), but have been described free catalytic and regulatory subunits. Regulation of CK2 is not a classic regulation through second messengers or phosphorylation, furthermore the catalytic subunit posses constitutive activity and it has been postulated that regulation could be done by changes in subcellular distribution due to interaction with different proteins from defined subcellular compartments. The protein chaperone grp94 (94 kDa-glucose regulated protein) belongs to the hsp90 family and is localized in the Endoplasmic Reticulum (ER) of all eukaryotic cells. Is constitutively expressed in absence of stress and is overexpressed under stress conditions as calcium depletion, virus infection, reducing agents etc. In the present work using techniques as Surface Plasmon Ressonance (SPR), Far Western and Pull down assays we have demonstrated that carboxy-terminal domain of grp94 interacts with the catalytic subunit of CK2 (CK2a) but not with the regulatory subunit or with the holoenzyme (a2b2). We mapped the interacting region using different mutants of CK2a, and we delimited the interacting domain to the cluster of lysines present in the helix aC(K74-77) on CK2a. This basic cluster is highly conserved in CK2a from different species but is not present in other kinases of the same family. Furthermore we demonstrated that grp94 has chaperone activity in vitro on CK2a and citrate synthase (CS). Light scattering of aggregated samples (aggregation promoted by heat shock) and electronic microscope imaging of aggregated samples have demonstrated that grp94 is able to protect CK2a and CS from aggregation. Chaperone activity depends on the maintenance of oligomeric state of grp94, then in reducing conditions when grp94 has lost its quaternary structure the ability to inhibit aggregation is diminished. In this work we also have been studied the subcellular distribution of CK2 and grp94 in an animal model of genetically obese rats (fa/fa). Zucker fa/fa rats are hyperinsulinemics and insulin resistant due to a down-regulation of insulin receptor. Additionally it has been described that in cultured cells insulin promoted the overexpression of grp94. In our model there is no variation of the levels or distribution of grp94 but exist differences in the levels and distribution of CK2 in liver of obese rats. In this rats CK2 is lower in the cytosolic fraction and increase in membranous fractions. |
| File Format | PDF HTM / HTML |
| Alternate Webpage(s) | https://ddd.uab.cat/pub/tesis/2002/tdx-1128102-175059/nra1de7.pdf |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
