Dephosphorylation Of CREB By PP1 And PP2A Regulates Its Tumorigenecity In Skin Cancer JB6 Cells

CREB（cAMP response element binding protein）is an important transcription factor in eukaryotic cells,and its transcriptional activity was first found to be regulated by its phosphorylation levels.CREB drives the expression of downstream genes through binding to cAMP response elements（CRE）,which is a conserved palindromic sequence TGACGTCA and activates downstream genes in the presence of cAMP.The CREB family proteins include CREB,CREM and ATF-1.They share similar domains and structures,with distinct critical phosphorylation sites in their kinase-inducible domains（KID）.Serine-133 is a crucial phosphorylation site for CREB.The phosphorylation of CREB is mainly regulated by signaling pathways such as cAMP pathway,MAPK pathway,Ras/Erk/RSK2 pathway,PLC^PKC signaling,PI3/AKT signaling and Ca²⁺-CaMK-CREB signaling.In this dissertation,I investigated the mechanisms of CREB-S133phosphorylation and dephosphorylation,and their association with cancer.In mouse epithelial cell line JB6,PKC activates PKA signaling,and also ERK1/2 and p38 pathways in MAPK signaling,to phosphorylate CREB-S133,in the presence of TPA.Among these kinases PKC,PKA,ERK1/2 are the main players.p38 also participates in the phosphorylation of CREB-133,however,not through direct binding to CREB.Furthermore,my results show that protein kinases JNKs and AKT are not directly involved in the phosphorylation of CREB-S133.Using in vitro inhibition through okadaic acid,dephosphoryla tion assay,in vivo knock-down of PP1 or PP2A catalytic subunits,and the Tet-On system to express PP-1βand PP-2Acαsubunits of PP1and PP2A,my work for the first time show the direct involvement of PP-1βand PP-2Acαin dephosphorylating CREB-S133.I further establi shed stable JB6 cell lines expressing wild type and mutant CREBs.Comparing their growth curve,cells with S133A-CREB,which mimics constitutive dephosphorylation,had the fastest growth rate,whereas cells with S133D-CREB,which mimics constitutive phosphorylation,had slower growth rate.Treating cells with okadaic acid followed by Hoechst staining show that cells expressing S133D-CREB had the highest apoptotic levels,whereas cells with S133A-CREB had lower apoptotic levels.Western blot analysis showed that S133D-CREB up-regulated the expression of tumor suppressor gene p53 and down-regulated the expression of Mcl-1,an anti-apoptotic gene.Soft agar colony formation experiments showed that cells with S133A-CREB form ed significantly more and larger colonies,whereas those with S133D-CREB formed fewer and smaller colonies.In addition,inhibiting protein kinases enhanced the colony formation of JB6 cells,whereas inhibiting the activity of phosphatases reduced their colony forma tion capacity.These results are the first to illustrate the associa tion between CREB phosphorylation levels and cell trans-formation.I further investigated the tumorigenecity of JB6 cells expressing either wild type or mutant CREB in nude mice.CREB dephosphorylation significantly enhanced its tumorigenecity.In conclusion,this thesis is the first to identify CREB-specific protein phosphatases in JB6 cells,and showed that the phosphorylation level of CREB is closely related to its tumorigenecity.These results shed new light on the progression of skin cancer,and provided new entry point for its diagnosis and therapeutic treatment.