Abstract
In response to epidermal growth factor (EGF) the microvillar core protein ezrin is phosphorylated transiently to a high level on tyrosine residues in human epidermoid carcinoma A431 cells. Here we report the identification of the tyrosine phosphorylation sites in ezrin using bacterially expressed protein as a substrate for in vitro phosphorylation with the EGF receptor. The two major phosphotyrosine-containing peptides observed in vivo were also phosphorylated in vitro. By secondary digestions and site-directed mutagenesis tyrosines 145 and 353 were identified as the sites of phosphorylation. One of the sites, Tyr145, lies in the N-terminal region of homology that is common to the band 4.1-talin-ezrin protein family. This tyrosine residue and its vicinal amino acids are conserved throughout the family members, including radixin, moesin, and the two phosphotyrosine phosphatases, PTP H1 and PTP MEG, but not in band 4.1 or talin. Tyr353 is localized within the alpha-helical domain of ezrin and comparison of the protein sequences reveals that this site is unique to ezrin.
Highlights
Localized within the a-helicadl omain of ezrin and com- transformed A431 cellsw, hich expressthe pp60""" and parison of the protein sequences reveals that this site P85gag-fepsrotein-tyrosine kinaserse, spectively [28, 29]
Neoplastic cell growth [1].The activation of growth factor stimulation of A431 cells causes the formationof microspikes receptor and nonreceptor protein-tyrosinekinases, and con- and microvilli leading to membraneruffling after 2-5 min and sequent phosphorylation of cytoplasmic substrates, initiates induces the rounding up of these cells
The identifi- epidermal growth factor (EGF) treatmentleads to an increase in tyrosine and threonine cation of receptor-like tyrosine phosphatases suggests that phosphorylation with kinetics that parallel the EGF-stimuthey may initiate signal transduction pathways in re- lated morphological changes [16,31].More than 10% of ezrin sponse to as yet unknown extracelsliuglnaarls [2,3]. is phosphorylated on tyrosine after treatment of A431 cells the number of these regulatory protein-tyrosine kinases and for 10 min with EGF [16], and the level of tyrosine phosphosphatases has been growing rapidly, the number of iden- phorylation may be even higher at earlier times [31]
Summary
Proteins were released ezrin cDNA [34] in Escherichia coli the vector set pRSET 6A, 6B, from the particles by boiling for 5 min in 1 X sample buffer and and 6C was used.' In short the pRSETvectors are derived from the analyzed on a 7.5% SDS-polyacrylamide gel as described previously. T o determine if a phosphopeptide is susceptible centrifugation the proteins were solubilized in 1X sample buffer (143 to cyanogen bromide (CNBr) cleavage, the 3'P-labeled fragments of mM dithiothreitol (DTT), 1%sodium dodecyl sulfate (SDS),10% ezrin were first cleaved with 5 pl of a 2.5 M CNBr solution dissolved glycerol, 63 mM Tris-C1, pH 6.8) by boiling and separated on a 15% in 70% formic acid by incubation overnight at room temperature and, SDS-polyacrylamide gel. The urea was removed by dialysis against 15 mM HEPES, pH 7.4, and the protein concentration adjusted to 0.3
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
