Phosphorylation of GFAP and lamin B and Astrocyte Activation as Revealed by Monoclonal Antibody J1-31




Ramsey, Gregory Ryan

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Monoclonal antibody Jl-31 has been shown to recognize cytoplasmic and nuclear antigens found in F98 glioblastoma cells in vitro and in astrocytes of the central nervous system near sites of injury. Jl-31 antigen(s) labeling increases in astrocytes and F98 cells when these cells are in a reactive state. Recent reports suggest that mAB Jl-31 specifically recognizes an epitope found on both GFAP and lamin B that contains a phosphorylated serine residue, and consensus sequences for appropriate protein kinases are present in GFAP and lamin B. To provide additional clues to the nature of the epitope recognized by mAB Jl-31 and to provide insight into signaling pathways involved in cytoskeletal remodeling associated with reactive astrocytes, F98 cells were activated in culture with forskolin and the resulting changes in intermediate filaments analyzed by immunocytochemical methods. Activation of adenylyl cyclase in F98 cells with forskolin resulted in a significant (p < 0.001) increase in mAB Jl-31 labeling of nuclear and cytoplasmic structures consistent with the known locations of lamins and GFAP. A similar increase in labeling intensity was not observed using antibodies specific for lamin B or GFAP. Further analysis included analysis of the Jl-31 antigens using immuno-magnetic bead precipitation from F98 cell lysates, and characterization of intracellular pathways involved in the forskolin induced increase in Jl-31 labeling intensity. Downstream targets of cAMP including PKA and Ca+2 channels were inhibited in an attempt to elucidate the kinase responsible for phosphorylating the Jl-31 antigen. Inhibitors of PKA had no effect on the forskolin induced increase in Jl-31 labeling, but treating F98 cells + 9 with verapamil, a known inhibitor of Ca channels, caused strong attenuation of the forskolin-induced increase in mAB Jl-31 labeling. These results support the notion that the Jl-31 mAB recognizes an epitope on GFAP and lamin B when that epitope contains a phosphorylated amino acid, most likely serine. Furthermore, that residue may be phosphorylated in a Ca+2 -dependent manner as a result of adenylyl cyclase activation. Immunoprécipitation of F98 cell extracts using magnetic beads with covalently bound mAB Jl-31 produced ambiguous results. Western analysis of proteins released from the beads identified GFAP and lamin B among others.



astrocytes, monoclonal antibodies, phosphorylation, antigens


Ramsey, G. R. (2005). Phosphorylation of GFAP and lamin B and astrocyte activation as revealed by monoclonal antibody J1-31 (Unpublished thesis). Texas State University-San Marcos, San Marcos, Texas.


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