Inhibition of NF-kappaB-dependent Bcl-xL expression by clusterin promotes albumin-induced tubular cell apoptosis.

Apoptosis and inflammation, important contributors to the progression of chronic kidney disease, can be influenced by clusterin (a secreted glycoprotein that regulates apoptosis) and nuclear factor-kappaB (NF-kappaB, a transcription factor modifying the expression of inflammatory genes). We studied proteinuria-induced renal disease and its influence on clusterin-mediated apoptosis. Exposure of cultured mouse proximal tubule epithelial cells to bovine serum albumin (BSA) resulted in activation of NF-kappaB and activator protein-1 (AP-1) within hours followed by a decline in their activation, decreased activation of extracellular signal-regulated kinases (ERK1/2), decreased cell-associated antiapoptotic Bcl-xL protein but increased apoptosis. Clusterin progressively increased in the media over a 3 day period. Clusterin siRNA blocked protein production, increased NF-kappaB activation, and significantly increased cellular Bcl-xL protein, thereby reducing spontaneous and BSA-induced apoptosis. An siRNA to the NF-kappaB inhibitor IkappaBalpha had similar results. BSA-stimulated NF-kappaB activation reciprocally decreased AP-1 activity by preventing ERK1/2 phosphorylation. These in vitro studies suggest that clusterin inhibits NF-kappaB-mediated antiapoptotic effects by the apparent stabilization of IkappaBalpha switching from promoting inflammation to apoptosis during proteinuria.

Proteolytic processing of the C terminus of the alpha(1C) subunit of L-type calcium channels and the role of a proline-rich domain in membrane tethering of proteolytic fragments.

Although most L-type calcium channel alpha(1C) subunits isolated from heart or brain are approximately 190-kDa proteins that lack approximately 50 kDa of the C terminus, the C-terminal domain is present in intact cells. To test the hypothesis that the C terminus is processed but remains functionally associated with the channels, expressed, full-length alpha(1C) subunits were cleaved in vitro by chymotrypsin to generate a 190-kDa C-terminal truncated protein and C-terminal fragments of 30-56 kDa. These hydrophilic C-terminal fragments remained membrane-associated. A C-terminal proline-rich domain (PRD) was identified as the mediator of membrane association. The alpha(1C) PRD bound to SH3 domains in Src, Lyn, Hck, and the channel beta(2) subunit. Mutant alpha(1C) subunits lacking either approximately 50 kDa of the C terminus or the PRD produced increased barium currents through the channels, demonstrating that these domains participate in the previously described (Wei, X., Neely, a., Lacerda, A. E. Olcese, r., Stefani, E., Perez-Reyes, E., and Birnbaumer, L. (1994) J. Biol. Chem. 269, 1635-1640) inhibition of channel function by the C terminus.

Identification of the sites phosphorylated by cyclic AMP-dependent protein kinase on the beta 2 subunit of L-type voltage-dependent calcium channels.

Voltage-dependent L-type calcium (Ca) channels are heteromultimeric proteins that are regulated through phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrated that the beta 2 subunit was a substrate for PKA in intact cardiac myocytes through back-phosphorylation experiments. In addition, a heterologously expressed rat beta 2a subunit was phosphorylated at two sites in vitro by purified PKA. This beta 2a subunit contains two potential consensus sites for PKA-mediated phosphorylation at Thr164 and Ser591. However, upon mutation of both of these residues to alanines, the beta 2a subunit remained a good substrate for PKA. The actual sites of phosphorylation on the beta 2a subunit were identified by phosphopeptide mapping and microsequencing. Phosphopeptide maps of a bacterially expressed beta 2a subunit demonstrated that this subunit was phosphorylated similarly to the beta 2 subunit isolated from heart tissue and that the phosphorylation sites were contained in the unique C-terminal region. Microsequencing identified three serine residues, each of which conformed to loose consensus sites for PKA-mediated phosphorylation. Mutation of these residues to alanines resulted in the loss of the PKA-mediated phosphorylation of the beta 2a subunit. The results suggest that phosphorylation of the beta 2a subunit by PKA occurs at three loose consensus sites for PKA in the C-terminus and not at either of the two strong consensus sites for PKA. The results also highlight the danger of assuming that consensus sites represent actual sites of phosphorylation. The actual sites of PKA-mediated phosphorylation are conserved in most beta 2 subunit isoforms and thus represent potential sites for regulation of channel activity. The sites phosphorylated by PKA are not substrates for protein kinase C (PKC), as the mutated beta 2 subunits lacking PKA sites remained good substrates for PKC.

Sorcin associates with the pore-forming subunit of voltage-dependent L-type Ca2+ channels.

Intracellular Ca2+ release in muscle is governed by functional communication between the voltage-dependent L-type Ca2+ channel and the intracellular Ca2+ release channel by processes that are incompletely understood. We previously showed that sorcin binds to cardiac Ca2+ release channel/ryanodine receptors and decreases channel open probability in planar lipid bilayers. In addition, we showed that sorcin antibody immunoprecipitates ryanodine receptors from metabolically labeled cardiac myocytes along with a second protein having a molecular weight similar to that of the alpha1 subunit of cardiac L-type Ca2+ channels. We now demonstrate that sorcin biochemically associates with cardiac and skeletal muscle L-type Ca2+ channels specifically within the cytoplasmically oriented C-terminal region of the alpha1 subunits, providing evidence that the second protein recovered by sorcin antibody from cardiac myocytes was the 240-kDa L-type Ca2+ channel alpha1 subunit. Anti-sorcin antibody immunoprecipitated full-length alpha1 subunits from cardiac myocytes, C2C12 myotubes, and transfected non-muscle cells expressing alpha1 subunits. In contrast, the anti-sorcin antibody did not immunoprecipitate C-terminal truncated forms of alpha1 subunits that were detected in myotubes. Recombinant sorcin bound to cardiac and skeletal HIS6-tagged alpha1 C termini immobilized on Ni2+ resin. Additionally, anti-sorcin antibody immunoprecipitated C-terminal fragments of the cardiac alpha1 subunit exogenously expressed in mammalian cells. The results identified a putative sorcin binding domain within the C terminus of the alpha1 subunit. These observations, along with the demonstration that sorcin accumulated substantially during physiological maturation of the excitation-contraction coupling apparatus in developing postnatal rat heart and differentiating C2C12 muscle cells, suggest that sorcin may mediate interchannel communication during excitation-contraction coupling in heart and skeletal muscle.

Identification and subcellular localization of the subunits of L-type calcium channels and adenylyl cyclase in cardiac myocytes.

The properties of cardiac L-type channels have been well characterized electrophysiologically, and many such studies have demonstrated that the channels are regulated by a cAMP-dependent pathway. However, the subunit composition of native cardiac L-type calcium channels has not been completely defined. Furthermore, a very important question exists regarding the status of the C-terminal domain of the pore-forming alpha1 subunit, as this domain has the potential to be the target of protein kinases but may be truncated as a result of post-translational processing. In the present studies, the alpha1C and beta2 subunits were identified by subunit-specific antibodies after partial purification from heart membranes, or immunoprecipitation from cardiac myocytes. Both the beta2 and the full-length alpha1C subunits were found to be expressed and co-localized in intact cardiac myocytes along T-tubule membranes. Using a quantitative antibody binding analysis, we demonstrated that the majority of the alpha1C subunits in intact cardiac myocytes appear to be full-length. In addition, we observed that adenylyl cyclase is localized in a pattern similar to the channel subunits in cardiac myocytes. Taken together, our results provide new insights into the structural basis for understanding the regulation of L-type calcium channels by a cAMP-mediated signaling pathway.

Differential effects of subunit interactions on protein kinase A- and C-mediated phosphorylation of L-type calcium channels.

We have expressed the pore-forming alpha1S (skeletal muscle isoform) and alpha1C (cardiac/brain isoform) subunits, as well as the accessory beta2a (cardiac/brain isoform) and alpha2/delta subunits of the L-type, dihydropyridine-sensitive calcium (Ca) channels in Spodoptera frugiperda insect cells (Sf9 cells) by infection with recombinant baculoviruses in order to facilitate biochemical studies of these rare, heteromultimeric membrane proteins. Since the L-type channels are believed to be regulated by protein phosphorylation, this expression system allowed us to investigate which subunits could act as substrates for protein kinase A and C (PKA and PKC) and to determine the potential role of subunit interactions in phosphorylation of the channel proteins. Using purified protein kinases in vitro, the membrane-associated alpha1S, alpha1C, and beta2a subunits were demonstrated to be phosphorylated stoichiometrically by PKA. The extent of phosphorylation of these subunits by PKA was similar whether the subunits were expressed alone or in combination. In addition, the alpha1C and beta2a subunits were phosphorylated stoichiometrically by PKC when expressed individually. In contrast, the alpha1S subunit, when expressed alone, was a poor substrate for PKC, despite the fact that this subunit has been shown to be an excellent substrate for PKC in native skeletal muscle membranes. Interestingly, co-expression of alpha1S with the beta2a subunit restored the ability of the alpha1S subunit to serve as a substrate for PKC. These results strongly suggests that subunit interactions play an important and potentially differential role in channel regulation by PKC, whereas phosphorylation of the same subunit by PKA occurs independent of subunit interaction. Furthermore, our results provide biochemical evidence that, when co-expressed, the alpha1C, alpha1S, and beta2a subunits of L-type Ca2+ channels are excellent substrates for PKA and PKC and support the hypothesis that phosphorylation of each of these subunits may participate in channel regulation by these kinases.

Structure and regulation of L-type calcium channels a current assessment of the properties and roles of channel subunits.

L-type Ca channels are complex heteromultimeric proteins that play important roles in the cardiovascular system. Recent studies have revealed new insights into how the pore-forming α(1) subunits interact with accessory subunits to produce functional Ca channels. The function of L-type Ca channels is often regulated by receptor-mediated signal transduction events that are thought to result in the phosphorylation of proteins that comprise the Ca channels. Although the molecular events underlying phosphorylation based regulation have been intensely investigated with the use of electrophysiological approaches, surprisingly few details are known about the biochemical events involved, and many questions remain unanswered. © 1996, Elsevier Science Inc. (Trends Cardiovasc Med 1996;6:265-273).

Roles of a membrane-localized beta subunit in the formation and targeting of functional L-type Ca2+ channels.

We report several unexpected findings that provide novel insights into the properties and interactions of the alpha 1 and beta subunits of dihydropyridine-sensitive L-type channels. First, the beta 2a subunit was expressed as multiple species of 68-72 kDa; the 70-72-kDa species arose from post-translational modification. Second, cell fractionation and immunocytochemical studies indicated that the hydrophilic beta 2a subunit, when expressed alone, was membrane-localized. Third, the beta 2a subunit increased the membrane localization of the alpha 1 subunit and the number of cells expressing L-type Ca2+ currents, without affecting the total amount of the expressed alpha 1C subunit. Expression of maximal currents in alpha 1C/beta 2a cotransfected cells paralleled the time course of expression of the beta subunit. Taken together, these results suggest that the beta subunit plays multiple roles in the formation, stabilization, targeting, and modulation of L-type channels.