PROTEIN TYROSINE KINASE SUBSTRATES IN RAT INTESTINAL MICROVILLUS MEMBRANES (MVM)

Abstract

Many growth factor receptors and proto-oncogenes regulate cellular proliferentiation and differentiation by an intrinsic tyrosine kinase which phosphorylates specific cellular substrates. These phosphotyrosyl proteins (p-tyr) are likely to be second messengers which transduce growth factor responses. To characterize these potential mediators of intestinal growth, we present the first identification of p-tyr in rat intestinal MVM, which we have previously shown to exhibit tyrosine kinase activity. (Gastroenterol 94:A460, 1988). MVM, purified from small intestine of fetal and adult rats by the calcium precipitation technique, or their corresponding intestinal homogenate, were incubated for 10 min at 20°C in vitro in Hepes/MnCl2ATP/Na orthovanadate. P-tyr were identified by either immunoprecipitation or Western blot using a highly specific anti-phosphotyrosine monoclonal or polyclonal antibody, respectively. A 68 kDa protein was the most abundant p-tyr identified in MVM (fetal > adult) using either technique. Immunoprecipitation demonstrated a 36 kDa p-tyr in fetal MVM and a 33 kDa p-tyr in adult MVM. A few other p-tyr were identified by Western blot in fetal MVM but only the 68 kDa was present in adult MVM. These p-tyr were significantly enriched in MVM compared to homogenates. To determine if these p-tyr are present in vivo and to study their developmental expression, Western blots were performed on MVM prepared from fetal, suckling and adult rat intestine in the presence of vanadate and NaF. The 68 kDa p-tyr was present in all ages studied and in MVM of adult jejunum and ileum. 80, 90 and 175 kDa p-tyr, previously identified in vitro, demonstrated variable expression during development. The epidermal growth factor receptor (EGFR), a known phosphotyrosyl protein, was immunoprecipitated from fetal MVM with an anti-EGFR antibody, incubated with 35P-ATP and identified as a single band of MW 175 kDa by autoradiography, suggesting that the 175 kDa p-tyr expressed in MVM is the EGFR. CONCLUSIONS: 1) MVM tyrosine kinase(s) phosphorylate p-tyr in vitro and in vivo. 2) A 68 kDa protein is the most adundant p-tyr present in fetal and adult MVM. 3) The 175 kDa p-tyr identified in MVM in vivo appears to be the EGFR which is autophosphorylated.