Phorbol dibutyrate-specific protein phosphorylation in brush border membranes of chicken enterocytes

We have demonstrated that phorbol esters such as phorbol dibutyrate (PhE) transiently inhibit Na/H exchange both in intact avian enterocytes and in brush border membrane (BBM) vesicles prepared from enterocytes treated with PhE (Chang et al., 1991, Am. J. Physiol. 260: C1264-C1272). Maximal inhibition occurs at 90 sec and values return to baseline by 15 min. In this study we examined if PhE causes changes in BBM protein phosphorylation by two methods: 1) in situ phosphorylation in which intact cells prelabeled with 32Pi were treated with PhE; 2) in vitro phosphorylation in which BBM, isolated from untreated and PhE-treated enterocytes, were exposed to gamma 32P-ATP. In situ phosphorylation studies showed that, at 90 sec, PhE increases the phosphorylation of BBM proteins of M(r) (pI): 150 (6.5), 89 (approximately 6.2), and 48 (approximately 6.1) kDa which declined to control values at 15 min, suggesting that these may be transport-related substrates. These labeled substrates were recovered in the detergent-insoluble fraction after extraction with 0.1% Triton X-100 overnight. Transient phosphorylation of a number of proteins was also observed when BBM prepared from control or PhE-treated cells were incubated with gamma 32P-ATP +/- 10 nM PhE, phosphatidyl serine, Ca2+, and/or exogenous protein kinase C (PKC). The in vitro phosphoproteins included both Triton-soluble and Triton-insoluble proteins. However, none of these proteins labeled in vitro coincided with those labeled in situ. The decline in phosphorylation with time can be accounted for by phosphatase action as these BBM possess a Ca-dependent phosphatase. In summary, we have demonstrated that the BBM possess PKC-specific substrates which can be visualized by in situ and in vitro phosphorylation. Treatment of intact enterocytes with PhE results in the phosphorylation of three detergent-insoluble proteins with a time course similar to that of PhE inhibition of Na/H transport.