Survival function of protein kinase C{iota} as a novel nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-activated bad kinase
Authors: Zhaohui Jin
Meiguo Xin
Xingming Deng
Journal: Journal of Biological Chemistry (2005)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: NNK; carcinogen; lung cancer cells; PKCiota; Bad phosphorylation
Affiliations: University of Florida Shands Cancer Center, Department of Medicine, University of Florida, Gainesville, Florida 32610-0232, USA.

Abstract:  Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is formed by nitrosation of nicotine and has been identified as the most potent carcinogen in cigarette smoke. NNK cannot only induce DNA damage but also promotes the survival of human lung cancer cells. Protein kinase C (PKC)iota is an atypical PKC isoform and plays an important role in cell survival, but the downstream survival substrate(s) is not yet identified. Bad, a proapoptotic BH3-only member of Bcl2 family, is co-expressed with PKCiota in both small cell lung cancer and non-small cell lung cancer cells. We discovered that NNK potently induces multisite Bad phosphorylation at Ser-112, Ser-136, and Ser-155 via activation of PKCiota in association with increased survival of human lung cancer cells. Purified, active PKCiota can directly phosphorylate both endogenous and recombinant Bad at these three sites and disrupt Bad/Bcl-XL binding in vitro. Overexpression of PKCiota results in an enhancement of Bad phosphorylation. NNK also stimulates activation of c-Src, which is a known PKCiota upstream kinase. Treatment of cells with the PKC inhibitor (staurosporine) or a Src-specific inhibitor (PP2) can block NNK-induced Bad phosphorylation and promote apoptotic cell death. The beta-adrenergic receptor inhibitor propranolol blocks both NNK-induced activation of PKCiota and Bad phosphorylation, indicating that NNK-induced Bad phosphorylation occurs at least in part through the upstream beta-adrenergic receptor. Mechanistically, NNK-induced Bad phosphorylation prevents its interaction with Bcl-XL. Because the specific depletion of PKCiota by RNA interference inhibits both NNK-induced Bad phosphorylation and survival, this confirms that PKCiota is a necessary component in NNK-mediated survival signaling. Collectively, these findings reveal a novel role for PKCiota as an NNK-activated physiological Bad kinase that can directly phosphorylate and inactivate this proapoptotic BH3-only protein, which leads to enhanced survival and chemoresistance of human lung cancer cells. 

Retracted: No

PubPeer comments:
 Figure 2C. Much more similar than you would expect. https: imgur.com hey49ly , 
 Images look similar, but these do not look like the same bands. , 
 #2 Dr. Deng: Iu2019m afraid I agree with the concerns in #1. I examined the image in inverse and by adjusting the brightness I can identify internal features that appear in both images. If necessary I can prepare a false color analysis to illustrate as well. *** ![file] , 
 #3 ![file] , 
 Thank you for your further information. I will ask my current employee to repeat this experiment after lab re-opening. I will revise this data after we finish the experiment. Thank you so much for your hard work. , 
 Correction: In Figure 2C, the Western bot of pBad (S155) in middle panel looks similar with pBad (S136). This may be a mistake. Now, corrected pBad (S155) is placed in Figure 2C, middle panel. This correction does not change any conclusions made in the article. We apologize for this error and any confusion it may have caused. We will contact the Journal to correct this accordingly. ![file] , 
 Correction: In Figure 2C, the Western bot of pBad (S155) in middle panel looks similar with pBad (S136). This may be a mistake. Now, corrected pBad (S155) is placed in Figure 2C, middle panel. This correction does not change any conclusions made in the article. We apologize for this error and any confusion it may have caused. We will contact the Journal to correct this accordingly. ![file] , 
 2020 correction. https: www.jbc.org content 295 42 14536.short There was a duplication in the loading control (u03b1-tubulin) between Figs. 1A and 4A. Fig. 4A was also incorrectly labeled. The new label matches Fig 1A. The figure legend for Fig. 4A should also read u201cWe repeated the use of the u03b1-tubulin loading control from Fig 1A.u201d For the middle panel of Fig. 2C, the immunobot of pBad (S155) was mistakenly duplicated with the same image of pBad (S136). The corrected immunoblot of pBad (S155) is placed in the revised Fig. 2C. In Fig. 8A, lanes 3 and 4 from immunoblots pBad (S112) and pBad (S136) look similar. The authors state that this is due to stripping and reprobing the same membrane sequentially with the two antibodies. These corrections do not change any conclusions made in the article. We apologize for any confusion the need for these corrections may have caused. ,