PMID- 32956670 OWN - NLM STAT- MEDLINE DCOM- 20211007 LR - 20211203 IS - 1095-8584 (Electronic) IS - 0022-2828 (Print) IS - 0022-2828 (Linking) VI - 149 DP - 2020 Dec TI - Inhibition of Pyk2 and Src activity improves Cx43 gap junction intercellular communication. PG - 27-40 LID - S0022-2828(20)30284-4 [pii] LID - 10.1016/j.yjmcc.2020.09.004 [doi] AB - Identification of proteins that interact with Cx43 has been instrumental in the understanding of gap junction (GJ) regulation. An in vitro phosphorylation screen identified that Protein tyrosine kinase 2 beta (Pyk2) phosphorylated purified Cx43CT and this led us to characterize the impact of this phosphorylation on Cx43 function. Mass spectrometry identified Pyk2 phosphorylates Cx43 residues Y247, Y265, Y267, and Y313. Western blot and immunofluorescence staining using HeLa(Cx43) cells, HEK 293 T cells, and neonatal rat ventricular myocytes (NRVMs) revealed Pyk2 can be activated by Src and active Pyk2 interacts with Cx43 at the plasma membrane. Overexpression of Pyk2 increases Cx43 phosphorylation and knock-down of Pyk2 decreases Cx43 phosphorylation, without affecting the level of active Src. In HeLa(Cx43) cells treated with PMA to activate Pyk2, a decrease in Cx43 GJ intercellular communication (GJIC) was observed when assayed by dye transfer. Moreover, PMA activation of Pyk2 could be inhibited by the small molecule PF4618433. This partially restored GJIC, and when paired with a Src inhibitor, returned GJIC to the no PMA control-level. The ability of Pyk2 and Src inhibitors to restore Cx43 function in the presence of PMA was also observed in NRVMs. Additionally, an animal model of myocardial infarction induced heart failure showed a higher level of active Pyk2 activity and increased interaction with Cx43 in ventricular myocytes. Src inhibitors have been used to reverse Cx43 remodeling and improve heart function after myocardial infarction; however, they alone could not fully restore proper Cx43 function. Our data suggest that Pyk2 may need to be inhibited, in addition to Src, to further (if not completely) reverse Cx43 remodeling and improve intercellular communication. CI - Copyright (c) 2020 Elsevier Ltd. All rights reserved. FAU - Zheng, Li AU - Zheng L AD - Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA. FAU - Trease, Andrew J AU - Trease AJ AD - Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA. FAU - Katsurada, Kenichi AU - Katsurada K AD - Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE 68198, USA. FAU - Spagnol, Gaelle AU - Spagnol G AD - Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA. FAU - Li, Hanjun AU - Li H AD - Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA. FAU - Shi, Wen AU - Shi W AD - Division of Cardiology, Department of Internal Medicine/Mary & Dick Holland Regenerative Medicine Program, University of Nebraska Medical Center, Omaha, NE 68198, USA. FAU - Duan, Bin AU - Duan B AD - Division of Cardiology, Department of Internal Medicine/Mary & Dick Holland Regenerative Medicine Program, University of Nebraska Medical Center, Omaha, NE 68198, USA. FAU - Patel, Kaushik P AU - Patel KP AD - Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE 68198, USA. FAU - Sorgen, Paul L AU - Sorgen PL AD - Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address: psorgen@unmc.edu. LA - eng GR - P30 GM106397/GM/NIGMS NIH HHS/United States GR - R01 GM072631/GM/NIGMS NIH HHS/United States GR - R01 GM131092/GM/NIGMS NIH HHS/United States GR - S10 RR027301/RR/NCRR NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20200918 PL - England TA - J Mol Cell Cardiol JT - Journal of molecular and cellular cardiology JID - 0262322 RN - 0 (Connexin 43) RN - EC 2.7.10.2 (Focal Adhesion Kinase 2) RN - EC 2.7.10.2 (src-Family Kinases) RN - NI40JAQ945 (Tetradecanoylphorbol Acetate) SB - IM MH - Amino Acid Sequence MH - Animals MH - Animals, Newborn MH - *Cell Communication MH - Cell Line MH - Connexin 43/chemistry/*metabolism MH - Disease Models, Animal MH - Focal Adhesion Kinase 2/*antagonists & inhibitors/metabolism MH - Gap Junctions/*metabolism MH - Heart Failure/enzymology/pathology MH - Heart Ventricles/pathology MH - Humans MH - Mutation/genetics MH - Phosphorylation MH - Protein Binding MH - Protein Domains MH - Rats MH - Tetradecanoylphorbol Acetate/pharmacology MH - src-Family Kinases/*antagonists & inhibitors/genetics/metabolism PMC - PMC7736531 MID - NIHMS1633709 OTO - NOTNLM OT - And gap junction OT - Cx43 OT - Phosphorylation OT - Pyk2 OT - Src COIS- Disclosures The authors declare that they have no conflicts of interest with the contents of this article. EDAT- 2020/09/22 06:00 MHDA- 2021/10/08 06:00 PMCR- 2021/12/01 CRDT- 2020/09/21 20:11 PHST- 2020/02/25 00:00 [received] PHST- 2020/09/08 00:00 [revised] PHST- 2020/09/10 00:00 [accepted] PHST- 2020/09/22 06:00 [pubmed] PHST- 2021/10/08 06:00 [medline] PHST- 2020/09/21 20:11 [entrez] PHST- 2021/12/01 00:00 [pmc-release] AID - S0022-2828(20)30284-4 [pii] AID - 10.1016/j.yjmcc.2020.09.004 [doi] PST - ppublish SO - J Mol Cell Cardiol. 2020 Dec;149:27-40. doi: 10.1016/j.yjmcc.2020.09.004. Epub 2020 Sep 18.