Protein kinase C mediated internalization of ErbB2 is independent of clathrin, ubiquitination and Hsp90 dissociation

Abstract

Overexpression of ErbB2 is frequent in cancer and understanding the mechanisms which regulate its expression is important. ErbB2 is considered endocytosis resistant. It has no identified ligand, but upon heterodimerization it is a potent mediator of proliferative signaling. A recent study established a role for protein kinase C (PKC) in internalization and recycling of ErbB2. We have now further investigated the molecular mechanisms involved in PKC-mediated downregulation of ErbB2. We confirm that PMA-induced PKC activation causes ErbB2 internalization, but while the Hsp90 inhibitor 17-AAG induced ErbB2 degradation, PMA had no such effect. When combined with 17-AAG, PMA had additive effect on ErbB2 internalization indicating that Hsp90 inhibition and PKC activation induce internalization by alternative mechanisms. We confirm that while 17-AAG-induced internalization was clathrin-mediated, PMA-induced internalization was clathrin independent. This difference may be explained by while both 17-AAG and PMA reduced the constitutive tyrosine phosphorylation of ErbB2, only 17-AAG induced Hsp90 dissociation, Hsp70 recruitment and ubiquitination of ErbB2. Importantly, since PMA induced internalization of ErbB2, but not dissociation of Hsp90, Hsp90 does not per se retain ErbB2 at the plasma membrane. The morphology of the compartment into which receptors are sorted upon PKC activation has not previously been identified. By immuno-electron microscopy, we show that PMA sorts ErbB2 into a complex tubulovesicular or cisternal organelle resembling a previously described endocytic recycling compartment.

Protein kinase C regulates ErbB3 turnover

“…This suggests that ligand-induced ubiquitination is a signal superior to the possible PKCinduced signal. This is in line with that PMA did not prevent 17-AAG induced degradation of ErbB2 (Bailey, Luan et al 2014, Dietrich, Malik et al 2018. Contrary to a previous study (Emkey and Kahn 1997), we did not observe any PMA-induced tyrosine phosphorylation of ErbB3.…”

“…In addition to constitutive and ligand-induced turnover, expression and localization of ErbB proteins are regulated through trans-activation by other receptors, both RTKs and GPCRs, and by other kinases like the PKCs. It has been shown for both EGFR (Bao, Alroy et al 2000, Llado, Timpson et al 2008, Liu, Idkowiak-Baldys et al 2013 and ErbB2 (Bailey, Luan et al 2014, Dietrich, Malik et al 2018) that PKC activation can induce their down-regulation from the plasma membrane and alter their intracellular sorting. In the current study we investigated and compared the effects of the PKC activator PMA, the PKC inhibitors Ro 31-8220 and Gö 6976, and siRNA-induced knockdown of PKCs, on ErbB3 localization and stability.…”

“…Turnover of ErbB proteins is to a large extent regulated by their internalization followed by either recycling to the plasma membrane, or sorting via early endosomes to late endosomes and lysosomes for degradation. Activation of PKCs has, however, been shown to inhibit the degradation of EGFR and ErbB2 (Bao, Alroy et al 2000, Llado, Timpson et al 2008, Idkowiak-Baldys, Baldys et al 2009, Liu, Idkowiak-Baldys et al 2013, Bailey, Luan et al 2014, Dietrich, Malik et al 2018). Since we and others have previously shown that ErbB3 is constitutively internalized and degraded with a short half-life (Waterman, Sabanai et al 1998, Cao, Wu et al 2007, Sak, Breen et al 2012, Fosdahl, Dietrich et al 2017, we first investigated the effect prolonged PKC-activation had on the total ErbB3 protein level.…”

“…Inhibition of either of these mechanisms can be expected to cause an increase in the level of ErbB3 at the plasma membrane. Activation of PKCs has on the other hand been shown to down-regulate EGFR and ErbB2 from the plasma membrane, without causing their subsequent degradation (Bao, Alroy et al 2000, Llado, Timpson et al 2008, Idkowiak-Baldys, Baldys et al 2009, Liu, Idkowiak-Baldys et al 2013, Bailey, Luan et al 2014, Dietrich, Malik et al 2018. Due to this we investigated the effect a prolonged PKC activation had on plasma membrane localized ErbB3.…”

“…It has previously been shown that PMA-induced activation of PKCs can cause down-regulation of EGFR and ErbB2 from the plasma membrane without leading to their subsequent degradation. The receptors are instead routed to and, to varying degree, sequestered in endosomes referred to as the endocytic recycling compartment (ERC) or the pericentrion (Bao, Alroy et al 2000, Llado, Timpson et al 2008, Idkowiak-Baldys, Baldys et al 2009, Liu, Idkowiak-Baldys et al 2013, Bailey, Luan et al 2014, Dietrich, Malik et al 2018. Contrary to what is reported for EGFR (Bao, Alroy et al 2000), down-regulation of ErbB2 appears to depend on its kinase activity (Bailey, Luan et al 2014).…”