Epidermal‐Growth‐Factor‐Dependent Activation of the Src‐Family Kinases

Abstract

The precise role of src-type kinases as signal transducers has been under intensive investigation but only in a few instances has their role been revealed in any detail. Thus, src, fyn and yes are activated upon stimulation by platelet-derived growth factor or colony-stimulating factor in cells expressing high levels of these receptors. Activation of src-family kinases by other receptor tyrosine kinases such as the epidermal-growth-factor (EGF) receptor has not been directly demonstrated. In this report, we demonstrate EGF-dependent activation of src-family tyrosine kinases in NIH3T3 cells overexpresssing the human EGF receptor. Activation is rapid (< 1 min) and persistent (up to 16 h). Furthermore, we show a correlation between the level of EGF receptor expressed and the degree of src-family kinase activation. We show that src-family kinase activity is also activated by addition of EGF to PC12 cells, which endogenously express relatively high levels of EGF receptor. Most strikingly, we show that A431 cells, which endogenously express very high levels of EGF receptor, show 10-fold elevated src-family kinase activity as compared to DHER14 cells, and that this activity is constitutive. This activity is completely blocked by AG1478, a specific inhibitor of the EGF-receptor tyrosine kinase activity, pointing to a direct link between overexpression of the EGF receptor and enhanced src-family kinase activity. Our findings suggest that EGF-dependent src-family kinase activity is detectable only when the levels of EGF receptor reach a specific level. Additionally, high levels of EGF receptor, as in A431 cells, may contribute to the elevated activation of src-family kinases. Sustained src-family kinase activation, similar to that seen in v-src-transformed cells, may play a role in tumorogenesis and tumor maintenance.The proto-oncoprotein pp60'-"" is a membrane-associated protein tyrosine kinase found in a wide variety of tissues and is a member of a group of at least nine non-receptor tyrosine kinases. Most of these src-type tyrosine kinases are expressed only in certain cell lines, but a few including src, fyn and yes are more widely expressed (reviewed in [l]).Although the exact role of these tyrosine kinases is still unclear, there is evidence for their involvement in signal transduction, mediating cell proliferation [2, 31, differentiation [4], and mitosis [l, 5, 6, 91.The mechanism of src regulation is complex and involves the action of inhibitory tyrosine kinases such as c-src kinase (CSK) [7], activating tyrosine phosphatases [8] and multiple serinekhreonine phosphorylations [9, 101. pp60"-'" kinase activity was shown to be stimulated upon entry into mitosis [5, 91, whereas the related fyn and lck are stimulated by the T-cell receptor following antigen stimulation (reviewed in [ll]). Lck is also activated upon CD4-CD8 crosslinking.The precise role of src-type kinases as signal transducers has been under intensive investigation but only in a few instances has their role been revealed in some detail. Thus, src, fyn ...

Pancreatic cancer cells require an EGF receptor-mediated autocrine pathway for proliferation in serum-free conditions

“…The expected observation that HPAF cells express binding sites for EGF and GRP is in agreement with the results of previous studies using other cell lines (Korc, 1991;Wang et al, 1996 Surprisingly, inhibiting the function of the EGF-receptor with an EGF receptor tyrosine kinase inhibitor, AG1478 (Osherov and Levitzki, 1994) but not a GRP receptor antagonist (RC3950-II) or an IGF-I receptor blocking antibody (αIR3) inhibited the proliferation of HPAF cells cultured continuously in serum free medium. AG1478 had no effect on IGF-1-stimulated proliferation in HPAF or PANC-1 cells; a further indication of the specificity of this agent for the EGF receptor tyrosine kinase (unpublished observations).…”

Growth inhibition of human lung adenocarcinoma cells by antibodies against epidermal growth factor receptor and by ganglioside GM3: involvement of receptor-directed protein tyrosine phosphatase(s)

“…The specific EGFR-blocking tyrphostins, AG 1478 and AG 1517, were kindly provided by Drs Gazit and Levitzki (Jerusalem, Israel), (Fry et al, 1994;Osherov and Levitzki, 1994;Levitzki and Gazit, 1995). They were dissolved in dimethyl sulphoxide (DMSO); for application in cell culture, the final DMSO concentration in the assays was < 0.…”

“…The relatively low potency of exogenously added EGF might be due to the constitutive presence of endogenous TGFa, which is known to be expressed in H125 cells (Soderdahl et al, 1988). In order to test whether an autocrine pathway involving the EGFR contributes to the growth of these cells, the effect of a recently discovered highly specific EGFR tyrosine kinase inhibitor, designated AG 1478 (Osherov and Levitzki, 1994;Levitzki and Gazit, 1995), was tested. H125 cells were cultured in the absence or presence of AG1478.…”

Signal‐Transduction Therapy

“…Thus, although mutated S K (v-src) has been demonstrated as an oncoprotein (pp60'-'" in chicken) its cellular homolog, the proto-oncoprotein pp60" transmits oncogenic signals of many receptors. Overexpression of EGFR or Her-2 in tumors leads to the constitutive activation of pp60'-"" (Lutrell et al, 1994;Osherov and Levitzki, 1994), where association between the RTK and c-Src may actually occur (Lutrell et al, 1994). Another possibility is that the Her-2 or EGFR to c-Src connection is mediated by the activation of a protein tyrosine phosphatase which removes the inhibitory phosphate from 51-527 (Osherov, 1994).…”