Biomedical subjects

Alan F Lau

Publications and source records attributed to Alan F Lau.

9 recordsLinked to original sources

PubMed2005

c-Src: bridging the gap between phosphorylation- and acidification-induced gap junction channel closure.

Gap junctions are a unique type of intercellular junction that mediate the direct exchange of small molecules between neighboring cells and play critical roles in the normal function of numerous organs. Mutations in the connexin proteins that make up gap junctions have been implicated in numerous human skin and neurosensory disorders. The ability of gap junctions to transmit molecules between cells is regulated by intracellular pH, the phosphorylation state of connexin, and the interaction of connexin with other cellular proteins. This Perspective focuses on the novel and complex events initiated by intracellular acidification resulting from tissue ischemia or hypoxia that lead to the interruption of intercellular communication between astrocytes. These events include alterations in connexin43 (Cx43) phosphorylation, disruption of beta-actin binding to Cx43, and the induced interaction of Cx43 with the c-Src tyrosine kinase, extracellular signal-regulated kinase 1 and 2, and mitogen-activated protein kinase phosphatase 1.

Animals↗

PubMed2005

Novel rab GAP-like protein, CIP85, interacts with connexin43 and induces its degradation.

Gap junctions play critical roles in tissue function and homeostasis. Connexin43 (Cx43) is a major gap junction protein expressed in the mammalian heart and other tissues and may be regulated by its interaction with other cellular proteins. Using the yeast two-hybrid screen, we identified a novel Cx43-interacting protein of 85-kDa, CIP85, which contains a single TBC, SH3, and RUN domain, in addition to a short coiled coil region. Homologues containing this unique combination of domains were found in human, D. melanogaster, and C. elegans. CIP85 mRNA is expressed ubiquitously in mouse and human tissues. In vitro interaction assays and in vivo co-immunoprecipitation experiments confirmed the interaction of endogenous CIP85 with Cx43. In vitro interaction experiments using CIP85 mutants with in-frame deletions of the TBC, SH3, and RUN domains indicated that the SH3 domain of CIP85 is involved in its interaction with Cx43. Conversely, analysis of Cx43 mutants with proline to alanine substitutions in the two proline-rich regions of Cx43 revealed that the P(253)LSP(256) motif is an important determinant of the ability of Cx43 to interact with CIP85. Laser-scanning confocal microscopy showed that CIP85 colocalized with Cx43 at the cell periphery, particularly in areas reminiscent of gap junction plaques. The functional importance of the interaction between CIP85 and Cx43 was suggested by the observation that CIP85 appears to induce the turnover of Cx43 through the lysosomal pathway.

Amino Acid Sequence↗

PubMed2004

Regulation of gap junctions by tyrosine protein kinases.

Most of the gap junction proteins are regulated in part by post-translational phosphorylation. Phosphorylation has been shown to be important in gap junction assembly and turnover, and for channel function in the resting state. Connexin phosphorylation may be altered by the activation of intracellular signaling pathways in response to growth factors, tumor promoters, activated oncogenes, hormones and inflammatory mediators. In some instances altered phosphorylation has been associated with changes in connexin function and in other cases appears to be associated with changes in the levels of the connexin protein and/or mRNA. This review focuses on the role of tyrosine protein kinases in the regulation of gap junctions. The literature is most extensive for connexin43 and those studies are reviewed here. A great deal has been learned in recent years about how connexin43 is regulated by tyrosine kinase-dependent signaling pathways. These pathways are often complex and to some extent are cell type- and stimulus-dependent. Although considerable progress has been made in unraveling the cellular pathways that regulate connexin function, significant challenges remain to be addressed in identifying additional phosphorylation sites and determining the stoichiometries of the phosphorylation events that regulate connexin function and it's interaction with other cellular proteins.

Animals↗

PubMed2004

The effects of connexin phosphorylation on gap junctional communication.

Gap junctions are specialized membrane domains composed of collections of channels that directly connect neighboring cells providing for the cell-to-cell diffusion of small molecules, including ions, amino acids, nucleotides, and second messengers. Vertebrate gap junctions are composed of proteins encoded by the "connexin" gene family. In most cases examined, connexins are modified post-translationally by phosphorylation. Phosphorylation has been implicated in the regulation of gap junctional communication at several stages of the connexin "lifecycle", such as the trafficking, assembly/disassembly, degradation, as well as, the gating of gap junction channels. Since connexin43 (Cx43) is widely expressed in tissues and cell lines, we understand the most about how it is regulated, and thus, connexin43 phosphorylation is a major focus of this review. Recent reports utilizing new methodologies combined with the latest genome information have shown that activation of several kinases including protein kinase A, protein kinase C, p34(cdc2)/cyclin B kinase, casein kinase 1, mitogen-activated protein (MAP) kinase and pp60(src) kinase can lead to phosphorylation at 12 of the 21 serine and two of the six tyrosine residues in the C-terminal region of connexin43. In several cases, use of site-directed mutants of these sites have shown that these specific phosphorylation events can be linked to changes in gap junctional communication.

Amino Acid Sequence↗

PubMed2002

Mechanism of v-Src- and mitogen-activated protein kinase-induced reduction of gap junction communication.

Connexin (Cx)43 gap junction channels are phosphorylated by numerous protein kinases, with the net effect typically being a reduction in gap junction communication (GJC). This reduction must result from a decrease in channel open probability, unitary conductance, or permselectivity, because previous results suggest that channel number is unaffected. Coexpression of v-Src with wild-type Cx43 (Cx43-wt) but not Cx43 with tyrosine to phenylalanine substitutions at 247 and 265 (Cx43-Y247,265F) resulted in reduced electrical and dye coupling but no change in single-channel amplitudes. EGF treatment of cells expressing Cx43-wt but not Cx43 with serine to alanine substitutions at 255, 279, and 282 (Cx43-S255,279,282A) resulted in reduced GJC, also with no change in single-channel amplitude. Dye coupling was reduced to a far greater extent than electrical coupling, suggesting that channel selectivity was also altered but with minimal effect on unitary conductance. The absence of Src- and MAPK-induced reductions in single-channel amplitude suggests that the decreases in GJC induced by these kinases result from reduced channel open probability and possibly altered selectivity.

Animals↗

PubMed2002

Connexin 43 expression in normal versus dysfunctional labor.

OBJECTIVE: We sought to determine whether connexin 43 (Cx43), the major myometrial gap junction protein, is differentially expressed in normal versus dysfunctional labor. STUDY DESIGN: Myometrial biopsies were obtained from 28 patients undergoing cesarean section and grouped into the following categories: (1) no labor, (2) dysfunctional labor, and (3) normal labor. Northern and Western analyses were performed to determine Cx43 messenger RNA (mRNA) and protein expression, respectively. Localization of Cx43 protein was determined by immunohistochemistry. RESULTS: Labor was associated with increased Cx43 mRNA expression (P <.05). This association was not true for protein. There was no difference in mRNA or protein expression between patients with normal labor versus those with dysfunctional labor. The extent of Cx43 immunohistochemical staining was not significantly different among the groups (P >.05). CONCLUSION: Dysfunctional labor is not associated with aberrant Cx43 mRNA or protein expression or with a reduction in immunodetectable Cx43 gap junctions.

Adult↗

PubMedDate not supplied

Connexin43 PDZ2 binding domain mutants create functional gap junctions and exhibit altered phosphorylation.

Connexin43 (Cx43) is the most abundantly expressed gap junction protein. The C-terminal tail of Cx43 is important for regulation of gap junctions via phosphorylation of specific tyrosine and serine residues and through interactions with cellular proteins. The C-terminus of Cx43 has been shown to interact with the PDZ2 domain of the tight and adherens junction associated zona occludens 1 (ZO-1) protein. Analysis of the PDZ2 binding domain of Cx43 indicated that positions -3 and -2, and the final hydrophobic amino acid at the C-terminus, are critical for ZO-1 binding. In addition, the C-termini of connexins 40 and 45, but not Cx32, interacted with ZO-1. To evaluate the functional significance of the Cx43-ZO-1 interaction, Cx43 wild type (Cx43wt) and mutants lacking either the C-terminal hydrophobic isoleucine (Cx43deltaI382) or the last five amino acids (Cx43delta378-382), required for ZO-1 binding in vitro, were introduced into a Cx43-deficient MDCK cell line. In vitro binding studies and coimmunoprecipitation assays indicated that these Cx43 mutants failed to interact with ZO-1. Confocal and deconvolution microscopy revealed that a fraction of Cx43wt colocalized with ZO-1 at the plasma membrane. A similar colocalization pattern was observed for the Cx43deltaI382 and Cx43 delta378-382 mutants, which were translocated to the plasma membrane and formed functional gap junction channels. The wt and mutant Cx43 appeared to have similar turnover rates. However, the P2 and P3 phosphoisoforms of the Cx43 mutants were significantly reduced compared to Cx43wt. These studies indicated that the interaction of Cx43 with ZO-1 may contribute to the regulation of Cx43 phosphorylation.

Animals↗

PubMedDate not supplied

v-Src tyrosine phosphorylation of connexin43: regulation of gap junction communication and effects on cell transformation.

The oncogenic tyrosine kinase, v-Src, phosphorylates connexin43 (Cx43) on Y247 and Y265 and inhibits Cx43 gap junctional communication (GJC), the process of intercellular exchange of ions and metabolites. To test the role of a negative charge on Cx43 induced by tyrosine phosphorylation, we expressed Cx43 with glutamic acid substitutions at Y247 or Y265. The Cx43Y247E or Cx43Y265E channels were functional in Cx43 knockout fibroblasts, indicating that introducing a negative charge on Cx43 was not likely the mechanism for v-Src disruption of GJC. Cells coexpressing v-Src and the triple serine to alanine mutant, Cx43S255/279/282A, confirmed that mitogen-activated protein (MAP) kinase phosphorylation of Cx43 was not required for v-Src-induced disruption of GJC and that tyrosine phosphorylation was sufficient. In addition, v-Src cells containing v-Src-resistant gap junctions, Cx43Y247/265F, displayed properties of cell migration, adhesion, and proliferation similar to Cx43wt/v-Src cells, suggesting that Cx43 tyrosine phosphorylation and disruption of GJC are not involved in these transformed cell properties.

Amino Acid Substitution↗

PubMedDate not supplied

Maintaining connexin43 gap junctional communication in v-Src cells does not alter growth properties associated with the transformed phenotype.

Loss of connexin expression and/or gap junctional communication (GJC) has been correlated with increased rates of cell growth in tumor cells compared to their normal communication-competent counterparts. Conversely, reduced rates of cell growth have been observed in tumor cells that are induced to express exogenous connexins and re-establish GJC. It is not clear how this putative growth-suppressive effect of the connexin proteins is mediated and some data has suggested that this function may be independent of GJC. In mammalian cells that express v-Src, connexin43 (Cx43) is phosphorylated on Tyr247 and Tyr265 and this results in a dramatic disruption of GJC. Cells that express a Cx43 mutant with phenylalanine mutations at these tyrosine sites form functional gap junctions that, unlike junctions formed by wild type Cx43, remain functional in cells that co-express v-Src. These cells still appear transformed; however, it is not known whether their ability to maintain GJC prevents the loss of growth restraints that confine "normal" cells, such as the inability to grow in an anchorage-independent manner or to form foci. In these studies, we have examined some of the growth properties of cells with Cx43 gap junctions that remain communication-competent in the presence of the co-expressed v-Src oncoprotein.

Animals↗