PHORBOL DIBUTYRATE-SPECIFIC PROTEIN-PHOSPHORYLATION IN BRUSH-BORDER MEMBRANES OF CHICKEN ENTEROCYTES

We have demonstrated that phorbol esters such as phorbol dibutyrate (P hE) 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-C127 2). 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 prot ein phosphorylation by two methods: 1) in situ phosphorylation in whic h intact cells prelabeled with P-32(i) were treated with PhE; 2) in vi tro phosphorylation in which BBM, isolated from untreated and PhE-trea ted enterocytes, were exposed to gamma(32)P-ATP. In situ phosphorylati on studies showed that, at 90 sec, PhE increases the phosphorylation o f BBM proteins of M(r) (pl): 150 (6.5), 89 (approximate to 6.2), and 4 8 (approximate to 6.1) kDa which declined to control values at 15 min, suggesting that these may be transport-related substrates. These labe led substrates were recovered in the detergent-insoluble fraction afte r extraction with 0.1% Triton X-100 overnight. Transient phosphorylati on of a number of proteins was also observed when BBM prepared from co ntrol or PhE-treated cells were incubated with gamma P-32-ATP +/- 10 n M PhE, phosphatidyl serine, Ca2+, and/or exogenous protein kinase C (P KC). The in vitro phosphoproteins included both Triton-soluble and Tri ton-insoluble proteins. However, none of these proteins labeled in vit ro coincided with those labeled in situ. The decline in phosphorylatio n with time can be accounted for by phosphatase action as these BBM po ssess a Ca-dependent phosphatase. In summary, we have demonstrated tha t the BBM possess PKC-specific substrates which can be visualized by i n situ and in vitro phosphorylation. Treatment of intact enterocytes w ith PhE results in the phosphorylation of three detergent-insoluble pr oteins with a time course similar to that of PhE inhibition of Na/H tr ansport. (C) 1994 Wiley-Liss, Inc.