Abstract. We show here that tubulin is the major in vivo substrate of the tyrosine-specific protein kinase pp60 ~-~ in nerve growth cone membranes. Phosphotyrosine antibodies were used to demonstrate phosphotyrosyl residues in a subpopulation of ~-and ~-tubulin that was highly enriched in a subcellular fraction of growth cone membranes from fetal rat brain. The presence of phosphotyrosine-modified isoforms of a-and ~-tubulin in vivo was confirmed by 32p labeling of rat cortical neurons in culture. Tubulin in growth cone membranes was phosphorylated at tyrosine in endogenous membrane phosphorylation reactions (0.068 mol phosphotyrosine/mol a-tubulin and 0.045 tool phosphotyrosine/mol/~-tubulin), and phosphorylation was specifically inhibited by antibodies directed against pp60 ~-s~, which is localized in the growth cone membranes, pp60 ~-s~ was capable of directly phosphorylating tubulin as shown in immune complex kinase assays with purified brain tubulin. Phosphopeptide mapping revealed a limited number of sites of tyrosine phosphorylation in a-and/~-tubulin, with similar phosphopeptides observed in vivo and in vitro. These results reveal a novel posttranslational modification of tubulin that could regulate microtubule dynamics at the growth cone.

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Lever pressing by rats (Rattus norvegicus) was maintained under a fixed-ratio 20 schedule of food presentation. Response rate-decreasing effects of the opioid compounds fentanyl, U50,488, butorphanol, and nalorphine were examined alone and in combination with the irreversible, ^.-selective opioid antagonist p-funaltrexamine (p-FNA). P-FNA antagonized the rate-decreasing effects of both fentanyl and butorphanol. p-FNA was more potent, and the duration of antagonism was greater, against .butorphanol than against fentanyl. [3-FNA also antagonized the effects of the higher nalorphine doses; however, lower doses of nalorphine, which were without effect alone, decreased response rates in the presence of p-FNA. The dose-effect curve for U50.488 was shifted leftward in the presence of p-FNA. These data suggest that p-FNA may be useful in assessing u.-receptor activity related to the effects of opioids on rate of operant behavior and the efficacy with which opioids produce these effects.Opioid antagonists are often used to make inferences about receptor mechanisms related to behavioral effects of opioid agonists, including effects on schedule-controlled operant behavior (e.g.,

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