Abstract
Phosphorylation of members of the connexin family of gap junction proteins has been correlated with gap junction assembly, but the mechanisms involved remain unclear. We have examined the role of casein kinase 1 (CK1) in connexin-43 (Cx43) gap junction assembly. Cellular co-immunoprecipitation experiments and in vitro CK1 phosphorylation reactions indicate that CK1 interacted with and phosphorylated Cx43, initially on serine(s) 325, 328, or 330. (32)P(i)-Metabolically labeled cells treated with CKI-7, a specific CK1 inhibitor, showed a reduction in Cx43 phosphorylation on site(s) that can be phosphorylated by CK1 in vitro. To examine CK1 function, normal rat kidney cells were treated with CKI-7, and Cx43 content was analyzed by Triton X-100 extraction, cell-surface biotinylation, and immunofluorescence. Western blot analysis indicated a slight increase in total Cx43, whereas gap junctional (Triton-insoluble) Cx43 decreased, and non-junctional plasma membrane Cx43 increased (as detected by cell surface biotinylation). Immunofluorescence experiments in the presence of CK1 inhibitor showed increases in Cx43 plasma membrane localization but not necessarily accumulation at cell-cell interfaces. Decreased gap junctional and phosphorylated Cx43 was also detected when cells were treated with IC261, a CK1 inhibitor specific for delta or epsilon isoforms. These data suggest CK1delta could regulate Cx43 gap junction assembly by directly phosphorylating Cx43.
Highlights
Gap junctional intercellular communication facilitates direct communication among adjacent cells by allowing passage of molecules less than 1000 Daltons [1, 2]
Cx43 Is Phosphorylated by casein kinase 1 (CK1)␦ in Vitro and in Cell Culture—Several studies have shown that Cx43 is phosphorylated on multiple different serine residues throughout its life cycle in homeostatic cells [12, 33, 34]
normal rat kidney (NRK) cells treated with CKI-7 for 2, 4, or 6 h were either directly solubilized in sample buffer to observe CKI-7 effects on total Cx43 (Fig. 4, Whole Cell panel) or were fractionated via Triton X-100 treatment into an insoluble fraction followed by immunoblot analysis for Cx43 (Fig. 4, Tritoninsoluble (Ins) panel)
Summary
Cx43, connexin-43; CK1, casein kinase 1; NRK, normal rat kidney; CT, carboxyl-terminal; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight mass spectrometry; NP, non-phosphorylated; GST, glutathione S-transferase; TM, triple mutant; PBS, phosphate-buffered saline; RIPA, radioimmune precipitation assay buffer; ZO-1, zona occludens 1. Recent reports link CK1, a constitutively active kinase, to cell cycle progression [18], nuclear protein translocation [19], intracellular protein trafficking [20], and cell morphogenesis [21]. CK1s are reported to be constitutively active, the CK1␦ and -⑀ isotypes have autoregulatory domains [22, 23]. CK1 activity plays a number of diverse biological roles and is critical for regulation of many cellular events. We have investigated the role of CK1 activity on Cx43 gap junction assembly and function in homeostatic NRK cells. Investigation of Cx43 trafficking after inhibition of CK1 function implies a role for CK1 activity in governing assembly of Cx43 gap junction channels
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