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![Casein kinase II can phosphorylate Ser 812 . A, immunoblot showing expression of endogenous casein kinase II (CK2) in lysates of mouse kidney and LLC-PK 1 cells by anti-CK2 antibody. CK2 protein serves as a positive control. B, schematic representation of GST fusion proteins used in in vitro CK2 phosphorylation analysis. C, in vitro phosphorylation by CK2 in the presence of [-32 P]ATP does not occur when the ERD is deleted (PC-2-15). Upper panel, autoradiography ; lower panel, immunoblot using anti-GST of the proteins used in the assay; the two samples shown were divided into equal aliquots and treated with () or without () CK2 in the upper panel experiments. D, in vitro phosphorylation by CK2 showing that Ser 812 is the substrate site. Upper panel, autoradiography ; lower panel, immunoblot with anti- GST of the same membrane showing equal loading of the S812A GST fusion protein that was not phosphorylated in the autoradiograph. All three lanes shown are from a single immunoblot.](profile/Qi-Qian-6/publication/8904406/figure/fig3/AS:339937791627275@1458058893795/Casein-kinase-II-can-phosphorylate-Ser-812-A-immunoblot-showing-expression-of.png)
Casein kinase II can phosphorylate Ser 812 . A, immunoblot showing expression of endogenous casein kinase II (CK2) in lysates of mouse kidney and LLC-PK 1 cells by anti-CK2 antibody. CK2 protein serves as a positive control. B, schematic representation of GST fusion proteins used in in vitro CK2 phosphorylation analysis. C, in vitro phosphorylation by CK2 in the presence of [-32 P]ATP does not occur when the ERD is deleted (PC-2-15). Upper panel, autoradiography ; lower panel, immunoblot using anti-GST of the proteins used in the assay; the two samples shown were divided into equal aliquots and treated with () or without () CK2 in the upper panel experiments. D, in vitro phosphorylation by CK2 showing that Ser 812 is the substrate site. Upper panel, autoradiography ; lower panel, immunoblot with anti- GST of the same membrane showing equal loading of the S812A GST fusion protein that was not phosphorylated in the autoradiograph. All three lanes shown are from a single immunoblot.
Source publication
Polycystin-2 (PC-2) is a non-selective cation channel that, when mutated, results in autosomal dominant polycystic kidney
disease. In an effort to understand the regulation of this channel, we investigated the role of protein phosphorylation in
PC-2 function. We demonstrated the direct incorporation of phosphate into PC-2 in cells and tissues and f...
Contexts in source publication
Context 1
... (CK2) (2), a ubiquitously distributed Ser/Thr protein kinase. To confirm that CK2 is expressed in renal epithelial cells where the poly- cystic kidney disease phenotype manifests, mouse kidney tis- sue as well as LLC-PK 1 epithelial cells were examined by immunoblotting using anti-CK2 antibody. CK2 is expressed in mouse kidney and LLC-PK 1 cells (Fig. 3A). We next tested whether PC-2 is phosphorylated by CK2 in vitro. Purified GST fusion proteins containing the COOH terminus of PC-2 with or without mutations affecting the phosphorylation sites (Fig. 3B) were assayed for phosphorylation by CK2. Deletion of the ER retention domain (ERD) that contains both Ser 801 and Ser 812 resulted in ...
Context 2
... as well as LLC-PK 1 epithelial cells were examined by immunoblotting using anti-CK2 antibody. CK2 is expressed in mouse kidney and LLC-PK 1 cells (Fig. 3A). We next tested whether PC-2 is phosphorylated by CK2 in vitro. Purified GST fusion proteins containing the COOH terminus of PC-2 with or without mutations affecting the phosphorylation sites (Fig. 3B) were assayed for phosphorylation by CK2. Deletion of the ER retention domain (ERD) that contains both Ser 801 and Ser 812 resulted in loss of phosphorylation by CK2 (Fig. 3C). When GST fusion proteins carrying single amino acid substitutions at Ser 801 and Ser 812 were assayed, S801A mutants were phospho- rylated normally, whereas S12A ...
Context 3
... PC-2 is phosphorylated by CK2 in vitro. Purified GST fusion proteins containing the COOH terminus of PC-2 with or without mutations affecting the phosphorylation sites (Fig. 3B) were assayed for phosphorylation by CK2. Deletion of the ER retention domain (ERD) that contains both Ser 801 and Ser 812 resulted in loss of phosphorylation by CK2 (Fig. 3C). When GST fusion proteins carrying single amino acid substitutions at Ser 801 and Ser 812 were assayed, S801A mutants were phospho- rylated normally, whereas S12A mutants were not (Fig. 3D). These finding are consistent with Ser 812 being a site of phos- phorylation by ...
Context 4
... for phosphorylation by CK2. Deletion of the ER retention domain (ERD) that contains both Ser 801 and Ser 812 resulted in loss of phosphorylation by CK2 (Fig. 3C). When GST fusion proteins carrying single amino acid substitutions at Ser 801 and Ser 812 were assayed, S801A mutants were phospho- rylated normally, whereas S12A mutants were not (Fig. 3D). These finding are consistent with Ser 812 being a site of phos- phorylation by ...
Citations
... A subsequent structure with the Nand C-termini of PC2 intact, including the regulatory C-terminal EF-hand motif (Ćelić et al., 2012;Yang et al., 2015), demonstrated a partially open conformation with cations in the ion conduction pathway (Wilkes et al., 2017). Since Ca 2+ ions have been shown to block the ion conduction pathway (Wilkes et al., 2017), it was speculated that this may reflect the regulatory role of Ca 2+ in the channel activity of PC2, whereby higher concentrations of cytosolic Ca 2+ inhibit channel activity (Cai et al., 2004). These structural studies suggest a role for the cytoplasmic regions in the regulation of PC2 channel activity. ...
... One possible means of reconciling these disparate data is to consider the subcellular distribution of PC2 and post-translational modifications to PC2. Studies on HEK, imCD3, and vascular smooth muscle preparations have indicated that PC2 can be post-translationally modified through acetylation, SUMOylation, and phosphorylation (Cai et al., 2004;Hasan et al., 2019;Hu & Harris, 2020;Padovano et al., 2017;Plotnikova et al., 2011). Specifically, SUMOylation protects PC2 from internalization into the plasma membrane of smooth muscle cells (Hasan et al., 2019). ...
Calcium release from the endoplasmic reticulum (ER) is predominantly driven by two key ion channel receptors, inositol 1, 4, 5‐triphosphate receptor (InsP3R) in non‐excitable cells and ryanodine receptor (RyR) in excitable and muscle‐based cells. These calcium transients can be modified by other less‐studied ion channels, including polycystin 2 (PC2), a member of the transient receptor potential (TRP) family. PC2 is found in various cell types and is evolutionarily conserved with paralogues ranging from single‐cell organisms to yeasts and mammals. Interest in the mammalian form of PC2 stems from its disease relevance, as mutations in the PKD2 gene, which encodes PC2, result in autosomal dominant polycystic kidney disease (ADPKD). This disease is characterized by renal and liver cysts, and cardiovascular extrarenal manifestations. However, in contrast to the well‐defined roles of many TRP channels, the role of PC2 remains unknown, as it has different subcellular locations, and the functional understanding of the channel in each location is still unclear. Recent structural and functional studies have shed light on this channel. Moreover, studies on cardiovascular tissues have demonstrated a diverse role of PC2 in these tissues compared to that in the kidney. We highlight recent advances in understanding the role of this channel in the cardiovascular system and discuss the functional relevance of PC2 in non‐renal cells. image
... Contrary to VOCCs, the ROCCs, in addition to Ca 2+ , carry K + , Na + , Ca 2+ , Ba 2+ , and Mg 2+ ions. Although some reports indicate that they are voltage-dependent, this aspect is still controversial [103][104][105][106][107][108][110][111][112][113][114][115][116][117][118][119][120]. Thus, they cannot be classified in the calcium-selective VOC category. ...
... These subfamilies are among the most abundant ion channels in the cardiovascular and nervous systems. TRPC1 and 2 (TRPP or PC2) channels have been well-studied in the last decade and have allowed us to describe some of their properties (Table 1) [103][104][105][106][107][108][110][111][112][113][114][115][116][117][118][119][120]. Single-channel recordings of native and expressed TRPC1 and TRPP2 in kidney insect SF9 and kidney HSG (or HEK-293) cell lines are very noisy and show an overlapping subconductance [107,113] which is difficult to differentiate between the different types and subtypes of TRP channels. ...
Calcium is a highly positively charged ionic species. It regulates all cell types’ functions and is an important second messenger that controls and triggers several mechanisms, including membrane stabilization, permeability, contraction, secretion, mitosis, intercellular communications, and in the activation of kinases and gene expression. Therefore, controlling calcium transport and its intracellular homeostasis in physiology leads to the healthy functioning of the biological system. However, abnormal extracellular and intracellular calcium homeostasis leads to cardiovascular, skeletal, immune, secretory diseases, and cancer. Therefore, the pharmacological control of calcium influx directly via calcium channels and exchangers and its outflow via calcium pumps and uptake by the ER/SR are crucial in treating calcium transport remodeling in pathology. Here, we mainly focused on selective calcium transporters and blockers in the cardiovascular system.
... Los primeros estudios sugerían que el canal era permeable a Ca 2+ (Hanaoka et al., 2000) o bloqueado por Ca 2+ (Cai et al., 2004), pero estudios recientes han mostrado consistentemente que el canal policistina-2 muestra una mayor preferencia hacia Na + y K + que hacia Ca 2+ (Shen et al., 2016;Liu et al., 2018). Es importante mencionar que existe evidencia que sugiere que la permeabilidad relativa a Ca 2+ podría variar en función de los canales o proteínas con las que tetrameriza policistina-2 (Mackay et al., 2020;Köttgen et al., 2008). ...
Este libro también busca dar visibilidad
a una familia muy grande y diversa de
proteínas conocidas como canales iónicos, las cuales participan activamente en
múltiples fenómenos celulares que son el
origen, por ejemplo, del latido cardiaco,
la contracción muscular, o diversos procesos sensoriales, entre otros. A través
de 13 capítulos, se describe el papel de
algunos canales iónicos y como su mal
funcionamiento o su expresión aberrante puede llevar a la aparición de diversas enfermedades. Aunque el número
de enfermedades asociadas a canales
iónicos crece año con año, en esta obra
nos enfocamos en dolencias de importancia mundial y nacional, como lo son
las arritmias cardiacas, las neurológicas
e inclusive el cáncer.
... CTDs of all three PKD2-related channel subunits have an EF-hand motif, followed by a coiled-coil domain and multiple intermittent serine phosphorylation sites. Phosphorylation of these sites enhances PKD2 current density in heterologous expression conditions, indicating this domain's role in channel membrane localization (58)(59)(60)(61). The contiguous cytoplasmic CTDs are not structurally determined in all reported PKD2 and PKD2L1 structures, but the EF-hand and coiled-coil structures have been solved as fragments (62)(63)(64)(65). ...
Polycystin subunits can form hetero- and homotetrameric ion channels in the membranes of various compartments of the cell. Homotetrameric polycystin channels are voltage- and calcium-modulated, whereas heterotetrameric versions are proposed to be ligand- or autoproteolytically regulated. Their importance is underscored by variants associated with autosomal dominant polycystic kidney disease and by vital roles in fertilization and embryonic development. The diversity in polycystin assembly and subcellular distribution allows for a multitude of sensory functions by this class of channels. In this review, we highlight their recent structural and functional characterization, which has provided a molecular blueprint to investigate the conformational changes required for channel opening in response to unique stimuli. We consider each polycystin channel type individually, discussing how they contribute to sensory cell biology, as well as their impact on the physiology of various tissues.
... PKD2 was found to be constitutively active on the plasma membrane of IMCD cells , while PKD2 heterologously expressed in other mammalian cells such as Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells, and Xenopus oocytes exhibited hardly detectable whole-cell currents, in part because of its relatively low density on the plasma membrane (Arif Pavel et al., 2016;Shen et al., 2016). Perhaps because of this, most functional characterizations of PKD2 were based on single-channel recordings (González-Perrett et al., 2001;Vassilev et al., 2001;Koulen et al., 2002;Cai et al., 2004) rather than measuring wholecell currents. Interestingly, both single-channel and whole-cilium currents mediated by PKD2 in renal primary cilia were detectable (Kleene and Kleene, 2017;Liu et al., 2018). ...
... Ca 2+ also exhibits complexed modulatory roles in PKD2: Sub-micromolar intracellular Ca 2+ activates PKD2 while high [Ca 2+ ], like [Mg 2+ ], inhibits the PKD2 channel activity. The phosphorylation at S812 by casein kinase II (CK2) was found to play an important role in this bell-shape dependence of the PKD2 function on the cytoplasmic [Ca 2+ ] (Cai et al., 2004). In fact, although PKD2 mutant S812A demonstrates similar bellshape dependence on the cytoplasmic [Ca 2+ ], it is 10-fold less sensitive to Ca 2+ than wide-type (WT) PKD2. ...
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the PKD1 or PKD2 gene which encodes membrane receptor PKD1 and cation channel PKD2, respectively. PKD2, also called transient receptor potential polycystin-2 (TRPP2), is a Ca ²⁺ -permeable channel located on the membrane of cell surface, primary cilia, and endoplasmic reticulum (ER). Ca ²⁺ is closely associated with diverse cellular functions. While ER Ca ²⁺ homeostasis depends on different Ca ²⁺ receptors, channels and transporters, the role of PKD2 within the ER remains controversial. Whether and how PKD2-mediated ER Ca ²⁺ leak relates to ADPKD pathogenesis is not well understood. Here, we reviewed current knowledge about the biophysical and physiological properties of PKD2 and how PKD2 contributes to ER Ca ²⁺ homeostasis.
... ER retention of PC2 depends on a 34-amino acid stretch between Glu787 and Ser820 in the carboxy terminus of the protein (Cai Table 5 Reported PKD2 mutations with consequences to PC2 channel function. al., 1999) that also contains two predicted phosphorylation sites, a protein kinase C (PKC) site at Ser801 and a casein kinase II site at Ser812 (Cai et al., 2004). The disease-causing truncated mutant R872X-PC2 also localized to the ER like the wild-type protein. ...
... The first phosphorylation site identified at the C-terminus of PC2 was identified in Ser812 (Cai et al., 2004), where dephosphorylation would reduce channel activation by intracellular Ca 2+ in the ER (Cai et al., 2004). This phosphorylation regulates PC2 trafficking (Köttgen et al., 2005) mediated by interaction with phosphofurine acid group classification proteins (PACS) PACS-1 and PACS-2 that are essential for retrograde traffic of PC2 between the plasma membrane, Golgi and ER. ...
... The first phosphorylation site identified at the C-terminus of PC2 was identified in Ser812 (Cai et al., 2004), where dephosphorylation would reduce channel activation by intracellular Ca 2+ in the ER (Cai et al., 2004). This phosphorylation regulates PC2 trafficking (Köttgen et al., 2005) mediated by interaction with phosphofurine acid group classification proteins (PACS) PACS-1 and PACS-2 that are essential for retrograde traffic of PC2 between the plasma membrane, Golgi and ER. ...
Polycystin-2 (TRPP2, PKD2, PC2) is the product of the PKD2 gene, whose mutations cause Autosomal Dominant Polycystic Kidney Disease (ADPKD). PC2 belongs to the superfamily of TRP (Transient Receptor Potential) proteins that generally function as Ca2+-permeable nonselective cation channels implicated in Ca2+ signaling. PC2 localizes to various cell domains with distinct functions that likely depend on interactions with specific channel partners. Functions include receptor-operated, nonselective cation channel activity in the plasma membrane, intracellular Ca2+ release channel activity in the endoplasmic reticulum (ER), and mechanosensitive channel activity in the primary cilium of renal epithelial cells. Here we summarize our current understanding of the properties of PC2 and how other transmembrane and cytosolic proteins modulate this activity, providing functional diversity and selective regulatory mechanisms to its role in the control of cellular Ca2+ homeostasis.
... Moreover, the inhibition of PDE1 or PDE3 increased proliferation of ADPKD cells. PDE1 inhibition triggered a mitogenic response to vasopressin in normal human kidney cells similar to the effect of restricting intracellular calcium (Cai et al., 2004;Cai et al., 2015;Pinto et al., 2016). Therefore, PDE1 may function as a link connecting changes in intracellular calcium and the activity of PDE3 pool controlling cell proliferation. ...
Cyclic nucleotide phosphodiesterases (PDEs) are superfamily of enzymes that regulate the spatial and temporal relationship of second messenger signaling in the cellular system. Among the 11 different families of PDEs, phosphodiesterase 1 (PDE1) sub-family of enzymes hydrolyze both 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) in a mutually competitive manner. The catalytic activity of PDE1 is stimulated by their binding to Ca2+/calmodulin (CaM), resulting in the integration of Ca2+ and cyclic nucleotide-mediated signaling in various diseases. The PDE1 family includes three subtypes, PDE1A, PDE1B and PDE1C, which differ for their relative affinities for cAMP and cGMP. These isoforms are differentially expressed throughout the body, including the cardiovascular, central nervous system and other organs. Thus, PDE1 enzymes play a critical role in the pathophysiology of diseases through the fundamental regulation of cAMP and cGMP signaling. This comprehensive review provides the current research on PDE1 and its potential utility as a therapeutic target in diseases including the cardiovascular, pulmonary, metabolic, neurocognitive, renal, cancers and possibly others.
... Phosphorylation of a Cterminal residue (S812) by protein kinase casein kinase 2 directs the channel to the ER, whereas its dephosphorylation by protein phosphatase A2 leads TRPP2 to the plasma membrane. 266,287 The C-terminal phosphorylation site is located within a cluster containing nine acidic residues that act as a PACS (phosphofurin acidic cluster sorting protein)-1 and À2 binding site. PACS interaction is enhanced upon phosphorylation and hence modulates cellular channel sorting. ...
The first genuine high-resolution single particle cryo-electron microscopy structure of a membrane protein determined was a transient receptor potential (TRP) ion channel, TRPV1, in 2013. This methodical breakthrough opened up a whole new world for structural biology and ion channel aficionados alike. TRP channels capture the imagination due to the sheer endless number of tasks they carry out in all aspects of animal physiology. To date, structures of at least one representative member of each of the six mammalian TRP channel subfamilies as well as of a few non-mammalian families have been determined. These structures were instrumental for a better understanding of TRP channel function and regulation. However, all of the TRP channel structures solved so far are incomplete since they miss important information about highly flexible regions found mostly in the channel N- and C-termini. These intrinsically disordered regions (IDRs) can represent between a quarter to almost half of the entire protein sequence and act as important recruitment hubs for lipids and regulatory proteins. Here, we analyze the currently available TRP channel structures with regard to the extent of these “missing” regions and compare these findings to disorder predictions. We discuss select examples of intra- and intermolecular crosstalk of TRP channel IDRs with proteins and lipids as well as the effect of splicing and post-translational modifications, to illuminate their importance for channel function and to complement the prevalently discussed structural biology of these versatile and fascinating proteins with their equally relevant ’unstructural’ biology.
... In the ER, TRPP2 can function as a Ca 2+ -activated channel permeable to Ca 2+ [138]. The open-state probability of TRPP2 follows a bell-shape Ca 2+ response with low levels of Ca 2+ (up to ~1 μM) increasing TRPP2 channel activity, whereas high Ca 2+ levels (≥1 μM) are inhibitory [139]. ...
Inside cells, the endoplasmic reticulum (ER) forms the largest Ca²⁺ store. Ca²⁺ is actively pumped by the SERCA pumps in the ER, where intraluminal Ca²⁺-binding proteins enable the accumulation of large amount of Ca²⁺. IP3 receptors and the ryanodine receptors mediate the release of Ca²⁺ in a controlled way, thereby evoking complex spatio-temporal signals in the cell. The steady state Ca²⁺ concentration in the ER of about 500 μM results from the balance between SERCA-mediated Ca²⁺ uptake and the passive leakage of Ca²⁺. The passive Ca²⁺ leak from the ER is often ignored, but can play an important physiological role, depending on the cellular context. Moreover, excessive Ca²⁺ leakage significantly lowers the amount of Ca²⁺ stored in the ER compared to normal conditions, thereby limiting the possibility to evoke Ca²⁺ signals and/or causing ER stress, leading to pathological consequences. The so-called Ca²⁺ leak channels responsible for Ca²⁺ leakage from the ER are however still not well understood, despite over 20 different proteins have been proposed to contribute to it. This review has the aim to critically evaluate the available evidence about the various channels potentially involved and to draw conclusions about their relative importance.
... The first phosphorylation site within the C-termini of PC2 was identified at Ser 812 and this remains the most studied residue [55]. Phosphorylation at this site is important for channel function; loss of phosphorylation resulted in reduced sensitivity to PC2 channel activation by intracellular Ca 2+ in ER-enriched liposomes [55]. ...
... The first phosphorylation site within the C-termini of PC2 was identified at Ser 812 and this remains the most studied residue [55]. Phosphorylation at this site is important for channel function; loss of phosphorylation resulted in reduced sensitivity to PC2 channel activation by intracellular Ca 2+ in ER-enriched liposomes [55]. A second study demonstrated a role for Ser 812 phosphorylation in regulating PC2 trafficking [56]. ...
... Although an initial study reported that PC2 was only phosphorylated at a single residue (Ser 812 ) [55], a second GSK3-recognition site was next identified at Ser 76 with a potential CK1 priming site at Ser 80 [61]. This phosphorylation event was required for PC2 trafficking to (or retention at) the lateral plasma membrane but not to primary cilia in kidney epithelial cells. ...
Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited cause of kidney failure and affects up to 12 million people worldwide. Germline mutations in two genes, PKD1 or PKD2, account for almost all patients with ADPKD. The ADPKD proteins, polycystin-1 (PC1) and polycystin-2 (PC2), are regulated by post-translational modifications (PTM), with phosphorylation, glycosylation and proteolytic cleavage being the best described changes. A few PTMs have been shown to regulate polycystin trafficking, signalling, localisation or stability and thus their physiological function. A key challenge for the future will be to elucidate the functional significance of all the individual PTMs reported to date. Finally, it is possible that site-specific mutations that disrupt PTM could contribute to cystogenesis although in the majority of cases, confirmatory evidence is awaited.
