Study On Targeting Of Protein Phoshpatases PP2A And PP2B To The C-Terminus Of The L-Type Calcium Channel Cav1.2

In the past ten years, cardiovascular and neurological diseases have been No.1 killers, which pose a grave threat to human health and lives. Not only are cardiovascular diseases the first cause of death in some developing countries, but they are also responsible for a rising tendency in morbidity and mortality. Currently, if there are three deaths around the world, one must relate to cardiovascular diseases. According to a lot of medical statistics, alternations of the function of the L-type calcium channel Cav1.2 are thought to be at the heart of several cardiovascular and neurological diseases. Cav1.2 is the most prominent channel in brain and heart[10]. It regulates brain activity and heartbeat. Ca2+ influx through the L-type channels is involved in the regulation of a variety of functions including muscle contraction, neurotransmitter release, hormone secretion, membrane excitability17-1, synaptic plasticity[1-4], gene expression[5,6], and etc. The L-type Ca2+ channel Cav1.2 constitutes a macromolecular signaling complex that comprises theβ2-adrenergic receptor, trimeric Gs protein, adenylyl cyclase, and cAMP-dependent protein kinase (PKA)[23,24] for efficient signaling cascades. Alterations of function of L-type Ca2+ channels have been implicated in a variety of cardiovascular diseases and neurological diseases, including arterial fibrillation, heart failure and ischemic neuronal cell death, etc.L-type Ca2+ channel plays an important role in cell functions, so it is not surprising that the channel is regulated by a lot of factors. In a typicalβ2 adrenergic receptor-cAMP-PKA signal transduction pathway, extracellular signaling molecules bind to theβ2 adrenergic receptor, activate trimeric Gs protein; activated Gs protein stimulates adenylyl cyclase and triggers an increase of the concentration of intracellular second message cAMP, which stimulates protein kinase A to phosphorylate its protein substrates. Protein phosphorylation and dephosphorylation, regulated by protein kinases and protein phosphatases, are involved in most physiological phenomena. As a result, as far as any specific signaling pathway is concerned, the activities of protein kinases and protein phosphatases must be precisely controlled relative to each other. Phosphatase PP2A regulates the phosphorylation state of Cav1.2 channel by dephosphorylating it, in oppose to the phosphorylating effect from PKA[11-12].In our previous work, we already defined that stimulation ofβ2. adrenergic receptor activates cAMP-PKA signal transduction pathway, which results in significant difference of Ca2+ current. PP2A counteracts the increase in Cav1.2 channel activity caused by PKA and other protein kinases, whereas PP2B can either augment or decrease Cav1.2 currents in cardiomyocytes depending on the precise experimental conditions. We found earlier that PP2A and PP2B are constitutively bound to Cav1.2[13.14] However, the interaction between Cav1.2 and PP2A or PP2B and functional consequence are not certain. This project is to investigate the association of the PP2A and PP2B with Cav1.2 and whether this association is critical for the regulation of Cav1.2 function.This study involves preparing a large amount of these purified proteins, and then conducting state-of-the-art protein biochemical experiments such as peptide array overlay, pull-down experiments of exogenous and co-immunoprecipitation of endogenous of PP2A with Cav1.2 to define and study protein interaction sites, electrophysiological recordings, and PP2A and PP2B activity assays to measure interaction of PP2A and PP2B with L-type calcium channel Cavl.2 during physiological activities. This work aims to elucidate the mechanism of targeting of protein phosphatases. The following parts describe the entire project.In the first part, GST-CT-8 encoding residues 1909-2029 of rabbit heart a11.2[13] served as a template for construction of GST-fusion proteins, covering residues 1909-1946 (CT-8-1), 1909-1971 (CT-8-2),1943-2029 (CT-8-3), and 1969-2029 (CT-8-4) and for a point mutation on the otherwise full-length GST-CT-8 construct to change Alal959 to Pro (GST-CT-8-P), as described[14]. GST-CT-B [15]ontaining residues 1694-1817 of rat a,1.2 cDNA (nearly identical to residues 1726-1849 of the above rabbit a1 1.2) was as given earlier. All plasmids should be transformed, purified, digested and sequenced. These GST-fusion proteins as well as the 6-His-PP2A/C[16] construct were expressed in Nova Blue (Novagen,Madison, WI) and BL21 Star (Invitrogen, San Diego, CA) E. coli strains and purified. PP2B was expressed in E. coli and purified[14,17].In the second part, firstly, The peptide spot array spanning residues 1784-2067 of rabbit cardiac al 1.2 (for sequence see GenBank accession number CAA33546) was synthesized on a PVDF membrane as published[18]. The first spot contains a 15-mer peptide covering residues 1784-1798 of al1.2. Peptides in each subsequent spot were shifted by one residue from the previous spot. The PVDF membrane was blocked before incubation with recombinant PP2A/C subunit expressed in Escherichia coli (see below) in the same solution, washed, and probed with the anti-PP2A/C antibody. Then, these GST-fusion proteins as well as the 6-His-PP2A/C and PP2B were used for in vitro pull-down interaction studies as detailed previously [14,16,19]. In the end, mouse (C57black/6) hearts and brains were homogenized in ice-cold homogenization buffers. Aliquots of the supernatants were incubated with the anti-Cavl.2 antibody or of nonspecific control IgG and prewashed protein A Sepharose slurry with Pep 1,4,5. Proteins were extracted by SDS sample buffer, separated by SDS-PAGE, transferred to PVDF membrane, and detected as above.In the third part, freshly prepared adult rabbit ventricular myocytes were preincubated, and Ca2+ current was recorded at room temperature in the whole cell perforated patch configuration using P-escin[20] Pipets were tip-filled with solution withβ-escin and either peptide 1,4, or no peptide added was used to backfill pipets to conduct electrophysiological analysis and test the change of current. In order to exclude the effect of Pepl,4,5 on phosphatase activity itself, PP2A catalytic activity was assessed using the DuoSet IC (R&D Systems) malachite green/molybdate-based PP2A activity assay [21,22] in accordance with the manufacturer's instructions. And PP2B/calcineurin activity was determined using the malachite green/molybdate-based calcineurin assay kit from Calbiochem following the manufacturer's instructions.In conclusion, we found that1. PP2A binds to two regions in the C-terminus of the central, pore-forming a subunit of Cav1.2:one region spans residues 1795-1818 and the other residues 1965-1971. PP2B binds immediately downstream of residue 1971 (1965LSPLLQR1971).2. Injection of a peptide (LSPLLQRSHSPTSLPRPCATPP) displaces PP2A from endogenous Cav1.2, but it does not displace PP2B from endogenous Cav1.2.3. Injection of a peptide (LSPLLQRSHSPTSLPRPCATPP) increases basal and isoproterenol-stimulated L-type Ca2+ currents in acutely isolated cardiomyocytes.4. Anchoring of PP2A at this site of Cav1.2 in the heart negatively regulates cardiac L-type currents, likely by counterbalancing phosphorylation mediated by PKA and possibly other kinases.