Abstract
Protein kinase Cs (PKCs) are activated by lipids in the plasma membrane and bind to a scaffold assembled on the epidermal growth factor (EGF) receptor (EGFR). Understanding how this complex is routed is important, because this determines whether EGFR is degraded, terminating signaling. Here, cells were preincubated in EGF-tagged gold nanoparticles, then allowed to internalize them in the presence or absence of a phorbol ester PKC activator. PKC colocalized with EGF-tagged nanoparticles within 5 min and migrated with EGFR-bearing vesicles into the cell. Two conformations of PKC-epsilon were distinguished by different primary antibodies. One, thought to be enzymatically active, was on endosomes and displayed a binding site for antibody RR (R&D). The other, recognized by Genetex green (GG), was soluble, on actin-rich structures, and loosely bound to vesicles. During a 15-min chase, EGF-tagged nanoparticles entered large, perinuclear structures. In phorbol ester-treated cells, vesicles bearing EGF-tagged nanoparticles tended to enter this endocytic recycling compartment (ERC) without the GG form. The correlation coefficient between the GG (inactive) and RR conformations on vesicles was also lower. Thus, active PKC has a Charon-like function, ferrying vesicles to the ERC, and inactivation counteracts this function. The advantage conferred on cells by aggregating vesicles in the ERC is unclear.
Highlights
IntroductionEpidermal growth factor (EGF) receptor (EGFR) is a member of a family of 170–185 KDa transmembrane receptors, known as the human epidermal growth factor (EGF) receptor (HER) or avian erythroblastosis oncogene B (ErbB) family
We found a persistent relationship of Protein kinase Cs (PKCs) with receptor-bearing vesicles, suggesting that PKC joined the EGFR at the plasma membrane and accompanied it during its travels
As a result that PKC-ε has an actin-binding motif at amino acids 223–228, we sought to determine whether actin binding was influencing enzyme location by comparing PKC to a protein whose actin-binding characteristics are well understood
Summary
Epidermal growth factor (EGF) receptor (EGFR) is a member of a family of 170–185 KDa transmembrane receptors, known as the human EGF receptor (HER) or avian erythroblastosis oncogene B (ErbB) family. Signaling from these receptors drives cell growth, so their mutation and overexpression promote tumor formation and serve as prognostic indicators of cancer progression. Drug resistance is often acquired through a mutation in the receptor itself or through changes that alter receptor processing so as to enhance signaling from the receptor (review [2]). Whereas many of the mechanisms that govern processing are known, the triggers that redirect receptor trafficking from one path to another are unknown. Does PKC regulate EGFR traffic?), we traced the co-internalization of EGFR and PKC
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