Expression and posttranslational modification of class III beta-tubulin during neuronal differentiation of P19...

We have used a combination of immunofluorescence microscopy, northern blotting, ELISA, and isoelectric focusing to characterize the expression of neuronal Class III beta-tubulin in P19 embryonal carcinoma cells induced to differentiate along a neuronal pathway by retinoic acid. Following 48 h differentiation, beta-III tubulin mRNA is evident and beta-III tubulin appears in the mitotic spindle of neuroblasts. Neurite outgrowth is obvious by day 3, and beta-III tubulin protein and mRNA levels increase concurrently until approximately day 7, when beta-III mRNA levels begin to decrease while protein levels remain high. In addition, increasingly acidic beta-III tubulin isoforms appear during neuronal differentiation. The expression of these isoelectric variants occurs concomitant with a temporal increase in the levels of beta-III tubulin present in the colchicine-stable microtubules. These results implicate posttranslational modifications of beta-III tubulin in the increased microtubule stability noted in differentiating P19 neurons.