Epidermal growth factor dependent phosphorylation of a 35-kilodalton protein in placental membranes.
In human placental membranes isolated in the presence of ethylenediaminetetraacetic acid (EDTA), epidermal growth factor (EGF) stimulated the [gamma-32P]ATP-dependent phosphorylation of tyrosine residues on the 170-kilodalton (kDa) EGF receptor and on a 35-kDa protein. The initial rate of phosphorylation of these proteins in the presence of EGF was 5.2 and 3.5 nmol of phosphate min-1 (mg of receptor protein)-1, and this was approximately 10- and 6-fold higher than the basal rate, respectively. Half-maximal phosphorylation of both proteins occurred at about 2.5 nM EGF. In the presence of p-nitrophenyl phosphate, EGF stimulated the phosphorylation of the 35-kDa protein but not the EGF receptor, suggesting that hormone-stimulated autophosphorylation of the receptor/kinase was not required for kinase activation. The 35-kDa protein exists in two forms: (1) 35Keluate, which was associated with the membrane in the presence of Ca2+ but was eluted with EDTA, and (2) 35Kmemb, which was not eluted from membranes with EDTA. Both forms were immunologically related to a 35-kDa protein previously isolated from A431 cells. Antiserum against the 35-kDa protein also reacted with a protein with an apparent size of 66 kDa that was phosphorylated in an EGF-dependent manner. In phosphorylation reactions performed in the presence of Mg2+, Ca2+ was required for phosphorylation of the 35Keluate form, but Ca2+ was not required for phosphorylation of the 35Kmemb form. Phosphorylation appears to change the membrane-binding properties of the 35Kmemb form because 32P-labeled 35Kmemb could be eluted from the membrane by EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)
D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005260:Female; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy
D008565:Membrane Proteins; D010750:Phosphoproteins; D004815:Epidermal Growth Factor; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D002118:Calcium