Connexin phosphorylation as a regulatory event linked to channel gating

The main proteins required for functional gap junction channels are known as connexins and most of their isoforms indicate that they can become phosphorylated. Connexin phosphorylation has been reported to participate in modifying junctional communication and the mechanisms involved apparently depend on which kinase becomes involved. Although multiple reports have suggested a strong influence of phosphorylation on channel gating, not enough physiological studies have been performed to determine precisely the gating mechanisms implicated. Moreover, gap junction channels follow other various gating mechanisms, including voltage gating and chemical gating, where phosphorylation could act as a modulator. The quest for this chapter has been to discriminate those instances where phosphorylation acts directly as a gating trigger and where it acts indirectly or only as a modulator. Despite recent efforts, the mechanisms involved in all these cases are barely understood.     

Connexin phosphorylation as a regulatory event linked to gap junction internalization and degradation

Gap junction proteins, connexins, are dynamic polytopic membrane proteins that exhibit unprecedented short half-lives of only a few hours. Consequently, it is well accepted that in addition to channel gating, gap junctional intercellular communication is regulated by connexin biosynthesis, transport and assembly as well as the formation and removal of gap junctions from the cell surface. At least nine members of the 20-member connexin family are known to be phosphorylated en route or during their assembly into gap junctions. For some connexins, notably Cx43, evidence exists that phosphorylation may trigger its internalization and degradation. In recent years it has become apparent that the mechanisms underlying the regulation of connexin turnover are quite complex with the identification of many connexin binding molecules, a multiplicity of protein kinases that phosphorylate connexins and the involvement of both lysosomal and proteasomal pathways in degrading connexins. This paper will review the evidence that connexin phosphorylation regulates, stimulates or triggers gap junction disassembly, internalization and degradation.     

Connexin phosphorylation as a regulatory event linked to gap junction channel assembly

Gap junctions, composed of proteins from the connexin family, allow for intercellular communication between cells and are important in development and maintenance of cell homeostasis. Phosphorylation has been implicated in the regulation of gap junctional communication at several stages of the cell cycle and the connexin “lifecycle”, such as trafficking, assembly/disassembly, degradation, as well as in the gating of “hemi” channels or intact gap junction channels. This review focuses on how phosphorylation can regulate the early stages of the connexin life cycle through assembly of functional gap junctional channels. The availability of sequences from the human genome databases has indicated that the number of connexins in the gene family is approximately 20, but we know mostly about how connexin43 (Cx43) is regulated. Recent technologies and investigations of interacting proteins have shown that activation of several kinases including protein kinase A, protein kinase C (PKC), p34^cdc2/cyclin B kinase, casein kinase 1 (CK1), mitogen-activated protein kinase (MAPK) and pp60^src kinase can lead to phosphorylation of the majority of the 21 serine and two of the tyrosine residues in the C-terminal region of Cx43. While many studies have correlated changes in kinase activity with changes in gap junctional communication, further research is needed to directly link specific phosphorylation events with changes in connexin oligomerization and gap junction assembly.     

Connexin phosphorylation as a regulatory event linked to gap junction internalization and degradation

Gap junction proteins, connexins, are dynamic polytopic membrane proteins that exhibit unprecedented short half-lives of only a few hours. Consequently, it is well accepted that in addition to channel gating, gap junctional intercellular communication is regulated by connexin biosynthesis, transport and assembly as well as the formation and removal of gap junctions from the cell surface. At least nine members of the 20-member connexin family are known to be phosphorylated en route or during their assembly into gap junctions. For some connexins, notably Cx43, evidence exists that phosphorylation may trigger its internalization and degradation. In recent years it has become apparent that the mechanisms underlying the regulation of connexin turnover are quite complex with the identification of many connexin binding molecules, a multiplicity of protein kinases that phosphorylate connexins and the involvement of both lysosomal and proteasomal pathways in degrading connexins. This paper will review the evidence that connexin phosphorylation regulates, stimulates or triggers gap junction disassembly, internalization and degradation.     

The Carboxyl Tail of Connexin32 Regulates Gap Junction Assembly in Human Prostate and Pancreatic Cancer Cells

Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size.     

Interplay between PKC and the MAP kinase pathway in Connexin43 phosphorylation and inhibition of gap junction intercellular communication

Gap junction channels are made of a family proteins called connexins. The best-studied type of connexin, Connexin43 (Cx43), is phosphorylated at several sites in its C-terminus. The tumor-promoting phorbol ester TPA strongly inhibits Cx43 gap junction channels. In this study we have investigated mechanisms involved in TPA-induced phosphorylation of Cx43 and inhibition of gap junction channels. The data show that TPA-induced inhibition of gap junction intercellular communication (GJIC) is dependent on both PKC and the MAP kinase pathway. The data suggest that PKC-induced activation of MAP kinase partly involves Src-independent trans-activation of the EGF receptor, and that TPA-induced shift in SDS-PAGE gel mobility of Cx43 is caused by MAP kinase phosphorylation, whereas phosphorylation of S368 by PKC does not alter gel migration of Cx43. We also show that TPA, in addition to phosphorylation of S368, also induces phosphorylation of S255 and S262, in a MAP kinase-dependent manner. The data add to our understanding of the molecular mechanisms involved in the interplay between signaling pathways in regulation of GJIC.     

Analysis of the distribution and phosphorylation state of ZO-1 in MDCK and nonepithelial cells

We have examined the distribution and extent of phosphorylation of the tight junction-associated protein ZO-1 in the epithelial MDCK cell line, and in three cell types that do not form tight junctions: S180 (sarcoma) cells, S180 cells transfected with E-cadherin (S180L), and primary cultures of astrocytes. In shortterm calcium chelation experiments on MDCK cells, removal of extracellular calcium caused cells to pull apart. However, ZO-1 remained concentrated at the plasma membrane and no change in ZO-1 phosphorylation was observed. Maintenance of MDCK cells in low calcium medium, conditions where no tight junctions are found, resulted in altered ZO-1 distribution and lower total phosphorylation of the protein. In S180 cells, ZO-1 was diffusely distributed along the entire cell surface, with concentration of the antigen in motile regions of the cell. Cell-cell contact was not a prerequisite for ZO-1 localization at the plasma membrane in this cell type, and the phosphate content of ZO-1 was found to be lower in S180 cells relative to MDCK cells. Expression of Ecadherin in S180L cells did not alter either the distribution or phosphorylation of ZO-1. In contrast to S180 cells, ZO-1 in primary cultures of astrocytes was concentrated at sites of cell-cell contact, and the phosphorylation state was the same as that in control MDCK cells. Comparison of one-dimensional proteolytic digests of ³²P-labeled ZO-1 revealed the phosphorylation of two peptides in control MDCK cells that was absent in both MDCK cells grown in low calcium and in S180 cells.We would like to thank Cheryl Richards for her help with the cell culture and immunohistochemistry; David Begg, Gary Firestone, Vik Maraj, Manijeh Pasdar and Colin Rasmussen for helpful discussions; Jaclyn Peebles and Greg Morrison for help with graphics and photography; and Grace Martin and Bob Campenot for rat tail collagen. We are grateful to all the members of our laboratories for their friendship, advice and support. This work was supported by an Establishment Award to B.R.S. from the Alberta Heritage Foundation for Medical Research and grants to B.R.S. from the Kidney Foundation of Canada and the Medical Research Council of Canada. A.H. is funded by a Studentship from the AHFMR. K.L.S. was supported by a grant from the National Institutes of Health (DK-42799) to Gary L. Firestone. B.R.S. is a Medical Research Council of Canada and AHFMR Scholar.     

Connexin 26 (GJB2) mutations, causing KID Syndrome, are associated with cell death due to calcium gating deregulation

The autosomic dominant KID Syndrome (MIM 148210), due to mutations in GJB2 (connexin 26, Cx26), is an ectodermal dysplasia with erythematous scaly skin lesions, keratitis and severe bilateral sensorineural deafness. The Cx26 protein is a component of gap junction channels in epithelia, including the cochlea, which coordinates the exchange of molecules and ions. Here, we demonstrate that different Cx26 mutants (Cx26D50N and Cx26G11E) cause cell death in vitro by the alteration of intra-cellular calcium concentrations. These results help to explain the pathogenesis of both the hearing and skin phenotypes, since calcium is also a potent regulator of the epidermal differentiation process.     

Sialic acids attached to O-glycans modulate voltage-gated potassium channel gating

Neuronal, cardiac, and skeletal muscle action potentials are produced and conducted through the highly regulated activity of several types of voltage-gated ion channels. Voltage-gated potassium (K(v)) channels are responsible for action potential repolarization. Glycans can be attached to glycoproteins through N- and O-linkages. Previous reports described the impact of N-glycans on voltage-gated ion channel function. Here, we show that sialic acids attached through O-linkages modulate gating of K(v)2.1, K(v)4.2, and K(v)4.3. The conductance-voltage (G-V) relationships for each isoform were shifted uniquely by a depolarizing 8-16 mV under conditions of reduced sialylation. The data indicate that sialic acids modulate K(v) channel activation through apparent electrostatic mechanisms that promote channel activity. Voltage-dependent steady-state inactivation was unaffected by changes in sialylation. N-Linked sialic acids cannot be responsible for the G-V shifts because K(v)4.2 and K(v)4.3 cannot be N-glycosylated, and immunoblot analysis confirmed K(v)2.1 is not N-glycosylated. Glycosidase gel shift analysis suggested that K(v)2.1, K(v)4.2, and K(v)4.3 were O-glycosylated and sialylated. To confirm this, azide-modified sugar residues involved specifically in O-glycan and sialic acid biosynthesis were shown to incorporate into all three K(v) channel isoforms using Cu(I)-catalyzed cycloaddition chemistry. Together, the data indicate that sialic acids attached to O-glycans uniquely modulate gating of three K(v) channel isoforms that are not N-glycosylated. These data provide the first evidence that external O-glycans, with core structures distinct from N-glycans in type and number of sugar residues, can modulate K(v) channel function and thereby contribute to changes in electrical signaling that result from regulated ion channel expression and/or O-glycosylation.     

Interpreting voltage-sensitivity of gap junctions as a mechanism of cardiac memory

Memory in the nervous system is essentially a network effect, resulting from activity-dependent synaptic modification in a network of neurons. Like the nervous system, the heart is a network of cardiac cells electrically coupled by gap junctions. The heart too has memory, termed cardiac memory, whereby the effect of an external electrical activation persists long after the presentation of stimulus is terminated. We have earlier proposed that adaptation of gap junctions, as a function of membrane voltages of the cells that are coupled by the gap junctions, is related to cardiac memory [V.S. Chakravarthy, J. Ghosh, On Hebbian-like adaption in heart muscle: a proposal for "Cardiac Memory", Biol. Cybern. 76 (1997) 207, J. Krishnan, V.S. Chakravarthy, S. Radhakrishnan, On the role of gap junctions on cardiac memory effect, Comput. Cardiol. 32 (2005) 13]. Using the proposed mechanism, we demonstrate memory effect using computational models of interacting cell pairs. In this paper, we address the biological validity of the proposed mechanism of gap junctional adaptation. It is known from electrophysiology of gap junctions that the conductance of these channels adapts as a function of junctional voltage. At a first sight, this form of voltage dependence seems to be at variance with the form required by our mechanism. But we show, with the help of a theoretical model, that the proposed mechanism of voltage-dependent adaptation of gap junctions, is compatible with the known voltage-sensitivity of gap junctions observed in electrophysiological studies. Our analysis suggests a new significance of the voltage-sensitivity of gap junctions and its possible link to the phenomenon of cardiac memory.     

Cyclin-cyclin-dependent kinase regulatory response is linked to substrate recognition

Cyclin/cyclin-dependent kinase (CDK) complexes are critical regulators of cellular proliferation. A complex network of regulatory mechanisms has evolved to control their activity, including activating and inactivating phosphorylation of the catalytic CDK subunit and inhibition through specific regulatory proteins. Primate herpesviruses, including the oncogenic Kaposi sarcoma herpesvirus, encode cyclin D homologues. Viral cyclins have diverged from their cellular progenitor in that they elicit holoenzyme activity independent of activating phosphorylation by the CDK-activating kinase and resistant to inhibition by CDK inhibitors. Using sequence comparison and site-directed mutagenesis, we performed molecular analysis of the cellular cyclin D and the Kaposi sarcoma herpesvirus-cyclin to delineate the molecular mechanisms behind their different behavior. This provides evidence that a surface recognized for its involvement in the docking of CIP/KIP inhibitors is required and sufficient to modulate cyclin-CDK response to a range of regulatory cues, including INK4 sensitivity and CDK-activating kinase dependence. Importantly, amino acids in this region are critically linked to substrate selection, suggesting that a mutational drift in this surface simultaneously affects function and regulation. Together our work provides novel insight into the molecular mechanisms governing cyclin-CDK function and regulation and defines the biological forces that may have driven evolution of viral cyclins.     

Forced gating motions by a substituted titratable side chain at the bundle crossing of a potassium channel

Numerous inwardly rectifying potassium (Kir) channels possess an aromatic residue in the helix bundle crossing region, forming the narrowest pore constriction in crystal structures. However, the role of the Kir channel bundle crossing as a functional gate remains uncertain. We report a unique phenotype of Kir6.2 channels mutated to encode glutamate at this position (F168E). Despite a prediction of four glutamates in close proximity, Kir6.2(F168E) channels are predominantly closed at physiological pH, whereas alkalization causes rapid and reversible channel activation. These findings suggest that F168E glutamates are uncharged at physiological pH but become deprotonated at alkaline pH, forcing channel opening due to mutual repulsion of nearby negatively charged side chains. The potassium channel pore scaffold likely brings these glutamates close together, causing a significant pK(a) shift relative to the free side chain (as seen in the KcsA selectivity filter). Alkalization also shifts the apparent ATP sensitivity of the channel, indicating that forced motion of the bundle crossing is coupled to the ATP-binding site and may resemble conformational changes involved in wild-type Kir6.2 gating. The study demonstrates a novel mechanism for engineering extrinsic control of channel gating by pH and shows that conformational changes in the bundle crossing region are involved in ligand-dependent gating of Kir channels.     

Capacitation-associated protein tyrosine phosphorylation starts early in buffalo (Bubalus bubalis) spermatozoa as compared to cattle

A comparative study was conducted on protein tyrosine phosphorylation events of capacitating sperm of two ruminant species, cattle and buffalo. Ejaculated cattle and buffalo bull spermatozoa were suspended separately in sp-TALP medium at 50 × 10⁶ mL⁻¹ and incubated at 38.5 °C with 5% CO₂ in air in the absence or presence of heparin for a period of 6 h. The extent of sperm capacitation after various periods of incubations was assessed by lysophosphatidyl choline-induced acrosome reaction followed by a triple-staining technique and capacitation-associated tyrosine-phosphorylated proteins were detected by immunoblotting technique using a monoclonal antiphosphotyrosine antibody. In the same media, over a time-period, a significant increase in capacitation percentage was observed even in control group of buffalo spermatozoa as compared to a non-significant increase in that of cattle sperm. In both cattle and buffalo spermatozoa, at 6 h, four proteins of molecular weight 49, 45, 32, and 20 kDa (designated as p49, p45, p32, and p20) were tyrosine phosphorylated. However, in buffalo, two additional proteins of 38 and 30 kDa were also tyrosine phosphorylated. In a time-course study, p20 appeared as early as at 0 h in capacitated buffalo spermatozoa as compared to 4 h in cattle. Further, in heparin-treated buffalo spermatozoa, with a time-dependent increase in tyrosine phosphorylation of some proteins, there was time-dependent dephosphorylation of some other proteins that was never seen in heparin-treated cattle sperm. Thus, the present findings revealed that though buffalo sperm takes more time than cattle for capacitation but its associated protein tyrosine phosphorylation event starts very early as compared to cattle.     

cAMP signaling regulates histone H3 phosphorylation and mitotic entry through a disruption of G2 progression

cAMP signaling is known to have significant effects on cell growth, either inhibitory or stimulatory depending on the cell type. Study of cAMP-induced growth inhibition in mammalian somatic cells has focused mainly on the combined role of protein kinase A (PKA) and mitogen-activated protein (MAP) kinases in regulation of progression through the G1 phase of the cell cycle. Here we show that cAMP signaling regulates histone H3 phosphorylation in a cell cycle-dependent fashion, increasing it in quiescent cells but dramatically reducing it in cycling cells. The latter is due to a rapid and dramatic loss of mitotic histone H3 phosphorylation caused by a disruption in G2 progression, as evidenced by the inhibition of mitotic entry and decreased activity of the CyclinB/Cdk1 kinase. The inhibition of G2 progression induced through cAMP signaling is dependent on expression of the catalytic subunit of PKA and is highly sensitive to intracellular cAMP concentration. The mechanism by which G2 progression is inhibited is independent of both DNA damage and MAP kinase signaling. Our results suggest that cAMP signaling activates a G2 checkpoint by a unique mechanism and provide new insight into normal cellular regulation of G2 progression.     

A Two-Dimensional Electrophoresis Study of Phosphorylation and Dephosphorylation of Chromaffin Cell Proteins in Response to a Secretory Stimulus

Phosphorylated proteins of bovine chromaffin cells, radioactively labeled with [32P]orthophosphate, have been analyzed by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Complex two-dimensional electrophoretograms were studied with the aid of computer-assisted image analysis (CAIA). A database map of 32P-labeled proteins was constructed; approximately 500 polypeptides have been detected, numbered, and characterized according to the intensity of labeling, molecular weight, and isoelectric point. The database was constructed from cells kept in resting conditions or stimulated with 59 mM K+ in 2.5 mM Ca2+ or in 0 Ca2+ solution. These manipulations caused statistically significant changes in the degree of phosphorylation of 20 proteins; they were classified as Ca2+-dependent substrates for the phosphorylation or dephosphorylation processes. These changes were also shown in cells stimulated in the presence of the Ca2+ channel activator Bay K 8644. New proteins that show as much as a fivefold increase in their phosphorylation state during cell stimulation have been located with this methodology, as well as many others that had not previously been detected with conventional methods. These experiments provide the first CAIA database of chromaffin cell phosphoproteins; the map constructed with these data will allow the location of specific phosphoproteins and serve as a reference for future ongoing studies. The database will continue to grow to identify more proteins and to facilitate the comparison of complex patterns obtained in different laboratories for normal and transformed pheochromocytoma PC12 cells.     

Plk3 functions as an essential component of the hypoxia regulatory pathway by direct phosphorylation of HIF-1alpha

Polo-like kinase 3 (Plk3) plays an important role in the regulation of cell cycle progression and stress responses. Plk3 also has a tumor-suppressing activity as aging PLK3-null mice develop tumors in multiple organs. The growth of highly vascularized tumors in PLK3-null mice suggests a role for Plk3 in angiogenesis and cellular responses to hypoxia. By studying primary isogenic murine embryonic fibroblasts, we tested the hypothesis that Plk3 functions as a component in the hypoxia signaling pathway. PLK3(-/-) murine embryonic fibroblasts contained an enhanced level of HIF-1α under hypoxic conditions. Immunoprecipitation and pulldown analyses revealed that Plk3 physically interacted with HIF-1α under hypoxia. Purified recombinant Plk3, but not a kinase-defective mutant, phosphorylated HIF-1α in vitro, resulting in a major mobility shift. Mass spectrometry identified two unique serine residues that were phosphorylated by Plk3. Moreover, ectopic expression followed by cycloheximide or pulse-chase treatment demonstrated that phospho-mutants exhibited a much longer half-life than the wild-type counterpart, strongly suggesting that Plk3 directly regulates HIF-1α stability in vivo. Combined, our study identifies Plk3 as a new and essential player in the regulation of the hypoxia signaling pathway.     

Tyrosine phosphorylation of DEPTOR functions as a molecular switch to activate mTOR signaling

Metabolic dysfunction is a major driver of tumorigenesis. The serine/threonine kinase mechanistic target of rapamycin (mTOR) constitutes a key central regulator of metabolic pathways promoting cancer cell proliferation and survival. mTOR activity is regulated by metabolic sensors as well as by numerous factors comprising the phosphatase and tensin homolog/PI3K/AKT canonical pathway, which are often mutated in cancer. However, some cancers displaying constitutively active mTOR do not carry alterations within this canonical pathway, suggesting alternative modes of mTOR regulation. Since DEPTOR, an endogenous inhibitor of mTOR, was previously found to modulate both mTOR complexes 1 and 2, we investigated the different post-translational modification that could affect its inhibitory function. We found that tyrosine (Tyr) 289 phosphorylation of DEPTOR impairs its interaction with mTOR, leading to increased mTOR activation. Using proximity biotinylation assays, we identified SYK (spleen tyrosine kinase) as a kinase involved in DEPTOR Tyr 289 phosphorylation in an ephrin (erythropoietin-producing hepatocellular carcinoma) receptor–dependent manner. Altogether, our work reveals that phosphorylation of Tyr 289 of DEPTOR represents a novel molecular switch involved in the regulation of both mTOR complex 1 and mTOR complex 2.     

Homeodomain-interacting protein kinase-2 stabilizes p27(kip1) by its phosphorylation at serine 10 and contributes to cell motility

HIPK2 is a serine/threonine kinase that acts as a coregulator of an increasing number of factors involved in cell survival and proliferation during development and in response to different types of stress. Here we report on a novel target of HIPK2, the cyclin-dependent kinase inhibitor p27(kip1). HIPK2 phosphorylates p27(kip1) in vitro and in vivo at serine 10, an event that accounts for 80% of the total p27(kip1) phosphorylation and plays a crucial role in the stability of the protein. Indeed, HIPK2 depletion by transient or stable RNA interference in tumor cells of different origin was consistently associated with strong reduction of p27(kip1) phosphorylation at serine 10 and of p27(kip1) stability. An initial evaluation of the functional relevance of this HIPK2-mediated regulation of p27(kip1) revealed a contribution to cell motility, rather than to cell proliferation, but only in cells that do not express wild-type p53.