C. Toskulkao et al., PHORBOL DIBUTYRATE-SPECIFIC PROTEIN-PHOSPHORYLATION IN BRUSH-BORDER MEMBRANES OF CHICKEN ENTEROCYTES, Journal of cellular physiology, 159(2), 1994, pp. 347-355
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.