The C-D interhelical domain of the serpin plasminogen activator inhibitor-type 2 is required for protection from TNF-alpha induced apoptosis.
The serine proteinase inhibitor (serpin), plasminogen activator inhibitor type 2 (PAI-2), has been reported to inhibit tumor necrosis factor-alpha (TNF) induced apoptosis. In order to begin to understand the molecular basis for this protection, we have investigated the importance of a structural domain within the PAI-2 molecule, the C-D interhelical region, in mediating the protective effect. The C-D interhelical region is a 33 amino acid insertion which is unique among serpins and has been implicated in transglutaminase catalyzed cross-linking of PAI-2 to cell membranes. We have constructed a mutant of PAI-2 wherein 23 amino acids are deleted from the C-D interhelical region generating a structure predicted to be homologous to the closely related, but non-inhibitory serpin, chicken ovalbumin. The PAI-2Delta65/87 deletion mutant retained inhibitory activity against its known serine proteinase target, urokinase-type plasminogen activator (uPA); however expression of this mutant in HeLa cells failed to protect from TNF-induced apoptosis. Analyses of the cellular distribution of PAI-2 showed that intracellular PAI-2, and not secreted or cell-surface PAI-2, was likely responsible for the observed protection from TNF-induced apoptosis. No evidence was found for specific cross-linking of PAI-2 to the plasma membrane in either control or TNF/cycloheximide treated cells. The data demonstrate that the PAI-2 C-D interhelical domain is functionally important in PAI-2 protection from TNF induced apoptosis and suggest a novel function for the C-D interhelical domain in the protective mechanism.
Dickinson
J L
JL
Queensland Cancer Fund Experimental Oncology Unit, The Queensland Institute of Medical Research, Brisbane, 4029, Australia.
Norris
B J
BJ
Jensen
P H
PH
Antalis
T M
TM
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D017124:Conserved Sequence; D017931:DNA Primers; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000502:physiology; D016296:Mutagenesis / Q000502:physiology; D017396:Plasminogen Activator Inhibitor 2 / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015843:Serpins / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D017931:DNA Primers; D017396:Plasminogen Activator Inhibitor 2; D015843:Serpins; D014409:Tumor Necrosis Factor-alpha