Voditelj:
Prof.dr.sc Hrvoje Banfić
Nakon preseljenja laboratorija u zgradu Hrvatskog instituta za istraživanje mozga nastavili smo s istraživanjem inozitol lipidnog signaliziranja u staničnoj jezgri. U modelu kompenzacijskog rasta jetre pokazali smo da nakon parcijalne hepatektomije dolazi do porasta fosfatidil-inozitol 3-fosfata u jezgri zbog aktivacije fosfoinozitol 3-kinaze C2 bete (1) koja se nalazi u jezgrinom matriksu (2) i koja se aktivira cijepanjem enzima pomoću kalpaina. Nadalje pokazali smo da u C2 domeni enzima postoji nuklearni lokalizacijski signal koji nužan za lokalizaciju enzima u staničnoj jezgri (3). U stanicama koje sinkrono prolaze kroz stanični ciklus enzim se aktivira tijekom G2/M faze staničnog ciklusa (4), dok diferencijacija stanica s pomoću sve-trans retinoične kiseline aktivira enzim putem tirozinske fosforilacije (5).
Također smo proučavali nazočnost i mehanizme aktivacije fosfolipaze C (PLC) u staničnoj jezgri. U modelu kompenzacijskog rasta jetre pokazali smo da su u jezgri nazočna tri izooblika. PLC-1b i PLC-1 nalaze se u jezgrinom matriksu, dok je izooblik PLC-1 nazočan u kromatinu (6). U HL-60 stanicama koje sinkrono prolaze kroz stanični ciklus enzim se aktivira na početku G1 faze, potom na kraju G1 faze, te konačno tijekom G2/M faze staničnog ciklusa. Uvijek se radi o PLC-1b izoobliku koji se aktivira fosforilacijom enzima na serinskom ostatku što je posredovano MEK kinazom (7, 8). S obzirom da se prilikom izolacije jezgara rabe deterđenti pa nije moguće u jezgrama određivati inozitolske fosfate zadnjih nekoliko godina počeli smo studirati njihov metabolizam u modelu pupajućeg kvasca Saccharomyces cerevisiae, jer je u kvascu mehanizam inozitolskog signaliziranja sličan onom u staničnim jezgrama (9). Inozitolski fosfati i pirofosfati su drugi glasnici koji nastaju postupnom fosforilacijom iz inozitol-1,4,5-trifosfata. Brojni radovi ukazuju na njihovu ulogu u regulaciji različitih staničnih procesa u eukariota, ali mnogi detalji su slabo poznati. Naša nedavna studija dokazala je genetsku, metaboličku i biokemijsku vezu da sinteza inozitolskih pirofosfata putem aktivacije PLC i enzima odgovornog za stvaranje inozitolskih pirofosfata (Kcs-1) igra važnu ulogu u signalnom slijedu neophodnom za progresiju staničnog ciklusa u kvascu sinkroniziranom s pomoću alfa-čimbenika (10). Međutim, nepoznato je koji je od inozitolskih pirofosfata odgovoran za taj učinak, te je isto tako nepoznato koja je veza između porasta inozitolskih pirofosfata putem aktivacije enzima Kcs-1 i progresije stanica kvasca kroz S fazu staničnog ciklusa. Cilj budućih istraživanja je razjasniti mehanizme putem kojih inozitolski pirofosfati reguliraju stanični ciklus. Uporabit ćemo niz delecijskih mutanti kvasca kako bi: a) pokazali koji je od inozitolskih pirofosfata odgovoran za prolaz stanica kroz S fazu staničnog ciklusa, b) testirali mogućnost da inozitolski pirofosfati koji se stvaraju u S fazi staničnog ciklusa reguliraju duljinu telomera, c) testirali mogućnost da je porast inozitolskih pirofosfata u stanicama sinkroniziranim s pomoću alfa-čimbenika odgovoran za pirofosforilaciju staničnih bjelančevina i/ili njihov izražaj odnosno modifikaciju i d) odredili promjene staničnog metabolizma u alfa-čimbenikom sinkroniziranim stanicama, te ih usporedili s promjenom razine inozitolskih pirofosfata.
Head:
Professor Hrvoje Banfić
After we have moved laboratory in the building of Croatian Institute for Brain Research we have continue to investigate mechanisms of inositol lipid signaling in the cell nuclei. In the model of compensatory liver hypertrophy we have shown that following partial hepatectomy there is increase in phosphatidylinositol 3-phosphate level in the liver nuclei as consequence of phosphoinositide 3-kinase C2beta activation (1) which is localized in the nuclear matrix (2) and is activated due to enzyme cleavage by calpain. Furthermore, we have shown that C2 domain of the enzyme contains nuclear localization signal which is crucial for nuclear localization of the enzyme (3). In synchronized cells the enzyme is activated during G2/M phase of the cell cycle (4), while in cells which are differentiated by all-trans-retinoic acid enzyme is activated via tyrosine phosphorylation (5).
Also we have investigated presence and mechanisms of activation of nuclear phospholipase C (PLC). In the model of compensatory liver growth we have shown that nuclei contains three isoforms of PLC. PLC-1b and PLC-1 are localized in the nuclear matrix while PLC-1 isoform is localized in the chromatin (6). In synchronized HL-60 cells the enzyme is activated at the beginning of G1 phase, then in the late G1 and finally during G/M phase of the cell cycle. The isoform which is activated is PLC-1b isoform and activation is due to serine phosphorylation caused by MEK kinase (7, 8). Because detergents are used during nuclei isolation and therefore inositol phosphate determination is impossible in the nuclei, in the last couple of years we have started to study inositol phosphate metabolism in budding yeast Saccharomyces cerevisiae, since in yeast inositol lipid signaling is similar to signaling in the cell nuclei (9). Inositol phosphates and pyrophosphates are second messengers generated by the sequential phosphorylation of inositol 1,4,5,-trisphosphate (InsP3). Several recent studies pointed to their role in the regulation of different cellular processes in eukaryotes, yet in many instances direct mechanistic roles remain elusive. Our recent study provided genetic, metabolic and biochemical evidence that synthesis of inositol pyrophosphates through activation of PLC and Kcs-1 play an important role in the signaling response required for cell cycle progression after mating pheromone arrest in the budding yeast (10). However, it is still uncertain which of the inositol pyrophosphates increased is/are responsible for Kcs-1-mediated effects, and it is unclear what might be the possible link between the Kcs-1-mediated increase in the level of pyrophosphates and the progression of yeast cells through the S phase of the cell cycle. The aim of the future research will be to define the mechanism of inositol pyrophosphates-mediated regulation of cell cycle progression. We will use a set of yeasts deletion mutants to define a) the particular pyrophosphate responsible for S phase-associated effects, b) to test for the possibility that inositol pyrophosphates generated during S phase progression regulate telomere length, c) to test the possibility that the increase in the level of pyrophosphates in alpha-factor synchronized cells is associated with an increase in pyrophosphorylation of the proteins and/or protein expression or modification, and d) to determine the changes in metabolism in alpha-factor synchronized cells and to correlate them with the changes in the level of inositol pyrophosphates.
- A. Sinđić, A. Aleksandrova, A.P. Fields, S. Volinia and H. Banfić: Presence and activation of nuclear phosphoinositide 3-kinase C2 during the compensatory liver growth. J. Biol. Chem. 276: 17754-17761, 2001.
- 48. A. Sinđić, V. Crljen, K. Matković, V. Lukinović-Škudar, D. Višnjić and H. Banfić: Activation of phosphoinositide 3-kinase C2 in the nuclear matrix during compensatory liver growth. Adv. Enzyme Regul. 46: 280-287, 2006.
- H. Banfić, D. Višnjić, N. Miše, S. Balakrishnan, S. Deplano, Y.E. Korchev and J. Domin: Epidermal growth factor stimulates translocation of the class II phosphoinositide 3-kinase PI3K-C2-beta to the nucleus. Biochem. J. 422: 53-60, 2009.
- D. Višnjić, J. Ćurić, V. Crljen, D. Batinić, S. Volinia and H. Banfić: Nuclear phosphoinositide 3-kinase C2 activation during G2/M phase of the cell cycle in HL-60 cells. Biochim. Biophys. Acta 1631: 61-71, 2003.
- D. Višnjić, V. Crljen, J. Ćurić, D. Batinić, S. Volinia and H. Banfić: The activation of nuclear phosphoinositide 3-kinase C2 in all-trans-retinoic acid-differentiated HL-60 cells. FEBS Lett. 529: 268-274, 2002.
- V. Crljen, D. Višnjić and H. Banfić: Presence of different phospholipase C isoforms in the nucleus and their activation during compensatory liver growth. FEBS Lett. 571: 35-42, 2004.
- V. Lukinović-Škudar, L. Đonlagić, H. Banfić and D. Višnjić: Nuclear phospholipase C- 1b activation during G2/M and late G1 phase in nocodazole –synchronized HL-60 cells. Biochim. Biophys. Acta, 1733: 148-156, 2005.
- V. Lukinović-Škudar, K. Matković, H. Banfić and D. Višnjić: Two waves of the nuclear phospholipase C activity in serum-stimulated HL-60 cells during G phase of the cell cycle. Biochim. Biophys. Acta 1771: 514-521, 2007.
- D. Višnjic and H. Banfić: Nuclear phospholipid signaling: phosphatidylinositol-specific phospholipase C and phosphoinositide 3-kinase. Pflugers Arch. 455: 19-30, 2007.
- H. Banfić, A. Bedalov, J. York and D. Višnjić: Inositol pyrophosphates modulate S phase progression after pheromone-induced arrest in Saccharomyces cerevisiae. J. Biol. Chem. 288: 1717-1725, 2013.
