Phosphorylation and the regulation of CD40L mRNA stability by polypyrimidine tract binding protein (P1394)

The expression of the CD40 ligand (CD40L) is controlled at the posttranscriptional level by an activation-induced program of regulated mRNA stability that is dependent on the formation of a cytoplasmic polypyrimidine tract binding protein (PTB)-containing-complex (Complex I) that binds to the 3’UTR of the CD40L mRNA. PTB is expressed as two major spliced isoforms in T cells, PTB-1 and PTB-4, which differ by the inclusion of exon 9 in PTB-4. To understand how these sites influence the function of PTB, mutants were generated at Ser 306 and Tyr 308 in the parent PTB-4 and introduced into Jurkat T cells expressing RNAi-mediated downregulated levels of PTB. Decreased PTB is known to result in an unstable CD40L transcript. We found that mutant proteins were expressed at relatively equal levels to each other and to that of exogenously-expressed wildtype PTB-4. Also, the distribution into the nuclear and cytoplasmic fractions was highly similar to endogenous PTB. We report the effect of the PTB mutations on the stability and cellular distribution of CD40L RNA. Also, comparing the functional activity of PTB-4 with PTB-1, which lacks the targeted phosphorylation sites, revealed distinct differences. Because we have previously demonstrated that PKC affects the PTB-mediated stability of CD40L mRNA, identifying the role of specific phosphorylation sites broadens our understanding of how events during T cell activation regulate expression of this critical co-stimulatory molecule.