Calphostin‐C stimulates epidermal growth factor receptor phosphorylation and internalization via light‐dependent mechanism

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Abstract
Calphostin‐C with perylenequinone structure is known to bind the regulatory domain of protein kinase C (PKC) and to inhibit kinase activity in vitro in a light‐dependent fashion. We have found that calphostin‐C induces substantial serine and threonine phosphorylation of the epidermal growth factor (EGF) receptor in a light‐dependent fashion in the EGF receptor‐hyperproducing squamous carcinoma cell line NA. Tryptic phospho‐peptide mapping and phospho‐amino acid analysis revealed that calphostin‐C–‐enhanced phosphorylation was on threonine 669, serine 671, serine 1046/1047, and serine 1166. However, caiphostin‐C did not inhibit phosphorylation of the 80 K protein, a cytosolic major substrate of PKC (MARCKS). Staurosporine, a potent PKC inhibitor with affinity for the catalytic domain of PKC, inhibited phosphorylation of the 80 K protein and 12‐O‐tetradecanoyl‐13‐phorbol acetate induction of EGF receptor phosphorylation but did not inhibit the calphostin‐C induction of the EGF receptor phosphorylation. These results suggest that the target of calphostin‐C in vivo is different from that of staurosporine and thus calphostin‐C in vivo does not inhibit PKC. Furthermore, calphostin‐C enhanced the internalization of phosphorylated EGF receptor. Thus, calphostin‐C apparently activates a novel signal transduction pathway which involves phosphorylation and internalization of the EGF receptor via light‐dependent mechanism.

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