Jacqueline Howie

Small Molecule Antagonists of the σ-1 Receptor Cause Selective Release of the Death Program in Tumor and Self-Reliant Cells and Inhibit Tumor Growth in Vitro and in Vivo

The acquisition of resistance to apoptosis, the cell’s intrinsic suicide program, is essential for cancers to arise and progress and is a major reason behind treatment failures. We show in this article that small molecule antagonists of the σ-1 receptor inhibit tumor cell survival to reveal caspase-dependent apoptosis. σ antagonist-mediated caspase activation and cell death are substantially attenuated by the prototypic σ-1 agonists (+)-SKF10,047 and (+)-pentazocine. Although several normal cell types such as fibroblasts, epithelial cells, and even σ receptor-rich neurons are resistant to the apoptotic effects of σ antagonists, cells that can promote autocrine survival such as lens epithelial and microvascular endothelial cells are as susceptible as tumor cells. Cellular susceptibility appears to correlate with differences in σ receptor coupling rather than levels of expression. In susceptible cells only, σ antagonists evoke a rapid rise in cytosolic calcium that is inhibited by σ-1 agonists. In at least some tumor cells, σ antagonists cause calcium-dependent activation of phospholipase C and concomitant calcium-independent inhibition of phosphatidylinositol 3′-kinase pathway signaling. Systemic administration of σ antagonists significantly inhibits the growth of evolving and established hormone-sensitive and hormone-insensitive mammary carcinoma xenografts, orthotopic prostate tumors, and p53-null lung carcinoma xenografts in immunocompromised mice in the absence of side effects. Release of a σ receptor-mediated brake on apoptosis may offer a new approach to cancer treatment.

AMP-activated protein kinase pathway: a potential therapeutic target in cardiometabolic disease.

AMPK (AMP-activated protein kinase) is a heterotrimetric enzyme that is expressed in many tissues, including the heart and vasculature, and plays a central role in the regulation of energy homoeostasis. It is activated in response to stresses that lead to an increase in the cellular AMP/ATP ratio caused either by inhibition of ATP production (i.e. anoxia or ischaemia) or by accelerating ATP consumption (i.e. muscle contraction or fasting). In the heart, AMPK activity increases during ischaemia and functions to sustain ATP, cardiac function and myocardial viability. There is increasing evidence that AMPK is implicated in the pathophysiology of cardiovascular and metabolic diseases. A principle mode of AMPK activation is phosphorylation by upstream kinases [e.g. LKB1 and CaMK (Ca2+/calmodulin-dependent protein kinase], which leads to direct effects on tissues and phosphorylation of various downstream kinases [e.g. eEF2 (eukaryotic elongation factor 2) kinase and p70 S6 kinase]. These upstream and downstream kinases of AMPK have fundamental roles in glucose metabolism, fatty acid oxidation, protein synthesis and tumour suppression; consequently, they have been implicated in cardiac ischaemia, arrhythmias and hypertrophy. Recent mechanistic studies have shown that AMPK has an important role in the mechanism of action of MF (metformin), TDZs (thiazolinediones) and statins. Increased understanding of the beneficial effects of AMPK activation provides the rationale for targeting AMPK in the development of new therapeutic strategies for cardiometabolic disease.

FXYD1 phosphorylation in vitro and in adult rat cardiac myocytes: threonine 69 is a novel substrate for protein kinase C

FXYD1 (phospholemman), the primary sarcolemmal kinase substrate in the heart, is a regulator of the cardiac sodium pump. We investigated phosphorylation of FXYD1 peptides by purified kinases using HPLC, mass spectrometry, and Edman sequencing, and FXYD1 phosphorylation in cultured adult rat ventricular myocytes treated with PKA and PKC agonists by phosphospecific immunoblotting. PKA phosphorylates serines 63 and 68 (S63 and S68) and PKC phosphorylates S63, S68, and a new site, threonine 69 (T69). In unstimulated myocytes, FXYD1 is approximately 30% phosphorylated at S63 and S68, but barely phosphorylated at T69. S63 and S68 are rapidly dephosphorylated following acute inhibition of PKC in unstimulated cells. Receptor-mediated PKC activation causes sustained phosphorylation of S63 and S68, but transient phosphorylation of T69. To characterize the effect of T69 phosphorylation on sodium pump function, we measured pump currents using whole cell voltage clamping of cultured adult rat ventricular myocytes with 50 mM sodium in the patch pipette. Activation of PKA or PKC increased pump currents (from 2.1 +/- 0.2 pA/pF in unstimulated cells to 2.9 +/- 0.1 pA/pF for PKA and 3.4 +/- 0.2 pA/pF for PKC). Following kinase activation, phosphorylated FXYD1 was coimmunoprecipitated with sodium pump alpha(1)-subunit. We conclude that T69 is a previously undescribed phosphorylation site in FXYD1. Acute T69 phosphorylation elicits stimulation of the sodium pump additional to that induced by S63 and S68 phosphorylation.

Regulation of the cardiac sodium pump.

In cardiac muscle, the sarcolemmal sodium/potassium ATPase is the principal quantitative means of active transport at the myocyte cell surface, and its activity is essential for maintaining the trans-sarcolemmal sodium gradient that drives ion exchange and transport processes that are critical for cardiac function. The 72-residue phosphoprotein phospholemman regulates the sodium pump in the heart: unphosphorylated phospholemman inhibits the pump, and phospholemman phosphorylation increases pump activity. Phospholemman is subject to a remarkable plethora of post-translational modifications for such a small protein: the combination of three phosphorylation sites, two palmitoylation sites, and one glutathionylation site means that phospholemman integrates multiple signaling events to control the cardiac sodium pump. Since misregulation of cytosolic sodium contributes to contractile and metabolic dysfunction during cardiac failure, a complete understanding of the mechanisms that control the cardiac sodium pump is vital. This review explores our current understanding of these mechanisms.

The inhibitory effect of phospholemman on the sodium pump requires its palmitoylation.

Background: Phospholemman regulates the plasmalemmal sodium pump in excitable tissues such as the heart. Results: Phospholemman is palmitoylated at two intracellular cysteines, and this reduces ion transport by the sodium pump. Conclusion: Phospholemman must be palmitoylated to inhibit the sodium pump. Significance: This is a potentially new way to regulate the sodium pump, an enzyme expressed in most eukaryotic cells. Phospholemman (PLM), the principal sarcolemmal substrate for protein kinases A and C in the heart, regulates the cardiac sodium pump. We investigated post-translational modifications of PLM additional to phosphorylation in adult rat ventricular myocytes (ARVM). LC-MS/MS of tryptically digested PLM immunoprecipitated from ARVM identified cysteine 40 as palmitoylated in some peptides, but no information was obtained regarding the palmitoylation status of cysteine 42. PLM palmitoylation was confirmed by immunoprecipitating PLM from ARVM loaded with [3H]palmitic acid and immunoblotting following streptavidin affinity purification from ARVM lysates subjected to fatty acyl biotin exchange. Mutagenesis identified both Cys-40 and Cys-42 of PLM as palmitoylated. Phosphorylation of PLM at serine 68 by PKA in ARVM or transiently transfected HEK cells increased its palmitoylation, but PKA activation did not increase the palmitoylation of S68A PLM-YFP in HEK cells. Wild type and unpalmitoylatable PLM-YFP were all correctly targeted to the cell surface membrane, but the half-life of unpalmitoylatable PLM was reduced compared with wild type. In cells stably expressing inducible PLM, PLM expression inhibited the sodium pump, but PLM did not inhibit the sodium pump when palmitoylation was inhibited. Hence, palmitoylation of PLM controls its turnover, and palmitoylated PLM inhibits the sodium pump. Surprisingly, phosphorylation of PLM enhances its palmitoylation, probably through the enhanced mobility of the phosphorylated intracellular domain increasing the accessibility of cysteines for the palmitoylating enzyme, with interesting theoretical implications. All FXYD proteins have conserved intracellular cysteines, so FXYD protein palmitoylation may be a universal means to regulate the sodium pump.

Substrate recognition by the cell surface palmitoyl transferase DHHC5.

Significance Dynamic palmitoylation at the cell surface by the acyl transferase DHHC5 regulates a plethora of physiological processes, from endocytosis to synaptic plasticity. Here we report that DHHC5 is abundantly expressed in cell surface caveolar microdomains in cardiac muscle, and that the Na pump regulator phospholemman is a substrate for DHHC5. Palmitoylation of phospholemman C40 by DHHC5 reduces Na pump activity. DHHC5 interacts with phospholemman independent of its PSD-95/Discs-large/ZO-1 homology (PDZ) binding motif, via a region of its intracellular carboxyl tail that has not hitherto been implicated in substrate recognition. This suggests that it may be possible to selectively manipulate DHHC5 activity by blocking the recruitment of specific substrates to the active site by the carboxyl tail. The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discs-large/ZO-1 homology (PDZ) binding motif, but requires a ∼120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme–substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipid interactions that inhibit the Na pump.

A Separate Pool of Cardiac Phospholemman That Does Not Regulate or Associate with the Sodium Pump: MULTIMERS OF PHOSPHOLEMMAN IN VENTRICULAR MUSCLE

Background: Phospholemman regulates the plasmalemmal sodium pump in excitable tissues. Results: In cardiac muscle, a subpopulation of phospholemman with a unique phosphorylation signature associates with other phospholemman molecules but not with the pump. Conclusion: Phospholemman oligomers exist in cardiac muscle. Significance: Much like phospholamban regulation of SERCA, phospholemman exists as both a sodium pump inhibiting monomer and an unassociated oligomer. Phospholemman (PLM), the principal quantitative sarcolemmal substrate for protein kinases A and C in the heart, regulates the cardiac sodium pump. Much like phospholamban, which regulates the related ATPase SERCA, PLM is reported to oligomerize. We investigated subpopulations of PLM in adult rat ventricular myocytes based on phosphorylation status. Co-immunoprecipitation identified two pools of PLM: one not associated with the sodium pump phosphorylated at Ser63 and one associated with the pump, both phosphorylated at Ser68 and unphosphorylated. Phosphorylation of PLM at Ser63 following activation of PKC did not abrogate association of PLM with the pump, so its failure to associate with the pump was not due to phosphorylation at this site. All pools of PLM co-localized to cell surface caveolin-enriched microdomains with sodium pump α subunits, despite the lack of caveolin-binding motif in PLM. Mass spectrometry analysis of phosphospecific immunoprecipitation reactions revealed no unique protein interactions for Ser63-phosphorylated PLM, and cross-linking reagents also failed to identify any partner proteins for this pool. In lysates from hearts of heterozygous transgenic animals expressing wild type and unphosphorylatable PLM, Ser63-phosphorylated PLM co-immunoprecipitated unphosphorylatable PLM, confirming the existence of PLM multimers. Dephosphorylation of the PLM multimer does not change sodium pump activity. Hence like phospholamban, PLM exists as a pump-inhibiting monomer and an unassociated oligomer. The distribution of different PLM phosphorylation states to different pools may be explained by their differential proximity to protein phosphatases rather than a direct effect of phosphorylation on PLM association with the pump.

Cardiac hypertrophy in mice expressing unphosphorylatable phospholemman

Aims Elevation of intracellular Na in the failing myocardium contributes to contractile dysfunction, the negative force–frequency relationship, and arrhythmias. Although phospholemman (PLM) is recognized to form the link between signalling pathways and Na/K pump activity, the possibility that defects in its regulation contribute to elevation of intracellular Na has not been investigated. Our aim was to test the hypothesis that the prevention of PLM phosphorylation in a PLM3SA knock-in mouse (in which PLM has been rendered unphosphorylatable) will exacerbate cardiac hypertrophy and cellular Na overload. Testing this hypothesis should determine whether changes in PLM phosphorylation are simply bystander effects or are causally involved in disease progression. Methods and results In wild-type (WT) mice, aortic constriction resulted in hypophosphorylation of PLM with no change in Na/K pump expression. This under-phosphorylation of PLM occurred at 3 days post-banding and was associated with a progressive decline in Na/K pump current and elevation of [Na]i. Echocardiography, morphometry, and pressure-volume (PV) catheterization confirmed remodelling, dilation, and contractile dysfunction, respectively. In PLM3SA mice, expression of Na/K ATPase was increased and PLM decreased such that net Na/K pump current under quiescent conditions was unchanged (cf. WT myocytes); [Na+]i was increased and forward-mode Na/Ca exchanger was reduced in paced PLM3SA myocytes. Cardiac hypertrophy and Na/K pump inhibition were significantly exacerbated in banded PLM3SA mice compared with banded WT. Conclusions Decreased phosphorylation of PLM reduces Na/K pump activity and exacerbates Na overload, contractile dysfunction, and adverse remodelling following aortic constriction in mice. This suggests a novel therapeutic target for the treatment of heart failure.

Palmitoylation of the Na/Ca exchanger cytoplasmic loop controls its inactivation and internalization during stress signaling

The electrogenic Na/Ca exchanger (NCX) mediates bidirectional Ca movements that are highly sensitive to changes of Na gradients in many cells. NCX1 is implicated in the pathogenesis of heart failure and a number of cardiac arrhythmias. We measured NCX1 palmitoylation using resin‐assisted capture, the subcellular location of yellow fluorescent protein‐NCX1 fusion proteins, and NCX1 currents using whole‐cell voltage clamping. Rat NCX1 is substantially palmitoylated in all tissues examined. Cysteine 739 in the NCX1 large intracellular loop is necessary and sufficient for NCX1 palmitoylation. Palmitoylation of NCX1 occurs in the Golgi and anchors the NCX1 large regulatory intracellular loop to membranes. Surprisingly, palmitoylation does not influence trafficking or localization of NCX1 to surface membranes, nor does it strongly affect the normal forward or reverse transport modes of NCX1. However, exchangers that cannot be palmitoylated do not inactivate normally (leading to substantial activity in conditions when wild‐type exchangers are inactive) and do not promote cargo‐dependent endocytosis that internalizes 50% of the cell surface following strong G‐protein activation or large Ca transients. The palmitoylated cysteine in NCX1 is found in all vertebrate and some invertebrate NCX homologs. Thus, NCX palmitoylation ubiquitously modulates Ca homeostasis and membrane domain function in cells that express NCX proteins.—Reilly, L., Howie, J., Wypijewski, K., Ashford, M. L. J., Hilgemann, D. W., Fuller, W. Palmitoylation of the Na/Ca exchanger cytoplasmic loop controls its inactivation and internalization during stress signaling. FASEB J. 29, 4532‐4543 (2015). www.fasebj.org

Regulation of the cardiac Na+ pump by palmitoylation of its catalytic and regulatory subunits

The Na+/K+-ATPase (Na+ pump) is the principal consumer of ATP in multicellular organisms. In the heart, the Na+ gradient established by the pump is essential for all aspects of cardiac function, and appropriate regulation of the cardiac Na+ pump is therefore crucial to match cardiac output to the physiological requirements of an organism. The cardiac pump is a multi-subunit enzyme, consisting of a catalytic α-subunit and regulatory β- and FXYD subunits. All three subunits may become palmitoylated, although the functional outcome of these palmitoylation events is incompletely characterized to date. Interestingly, both β- and FXYD subunits may be palmitoylated or glutathionylated at the same cysteine residues. These competing chemically distinct post-translational modifications may mediate functionally different effects on the cardiac pump. In the present article, we review the cellular events that control the balance between these modifications, and discuss the likely functional effects of pump subunit palmitoylation.