PRKX, a phylogenetically and functionally distinct cAMP-dependent protein kinase, activates renal epithelial cell migration and morphogenesis

Li, Xiaohong; Li, Hsi-Ping; Amsler, Kurt; Hyink, Deborah; Wilson, Patricia D.; Burrow, Christopher R.

doi:10.1073/pnas.132051799

Cited by 48 publications

(78 citation statements)

References 48 publications

(47 reference statements)

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“…The current observation that ␣ v ␤ 3 disengagement is associated with the accumulation of cyclic AMP in HK-2 cells and that inhibiting cyclic AMP signaling pathway impedes HK-2 cell migration provides strong evidence for the hypothesis that cyclic AMP signaling pathway plays a role in stimulating cell mobility in response to extracellular -calpain. Previous studies have also demonstrated that cyclic AMP signaling pathway stimulates tubular epithelial cell migration in vitro (28,29). In addition, cyclic AMP signaling pathway has been implicated in the protection of these cells against the cytotoxic effect of ischemia and intracellular ATP depletion (40).…”

Section: Discussionmentioning

confidence: 97%

“…Unligated ␣ v ␤ 3 Integrin Induces PKA-dependent Mobility of Proximal Tubular Epithelial Cells-Integrin ligation is a potent regulator of PKA, and in turn, PKA pathway regulates actinbased migration of cells (26,27), including tubular epithelial cells (28,29). Thus, we tested the hypothesis that the cyclic AMP/PKA pathway would become activated following calpaindependent disengagement of ␣ v ␤ 3 and was involved in HK-2 cell migration.…”

Section: Extracellular -Calpain Modulates the Behavior Of Proximalmentioning

confidence: 99%

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Extracellular Calpains Increase Tubular Epithelial Cell Mobility

Frangié¹,

Zhang

Perez

et al. 2006

Journal of Biological Chemistry

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Calpains are intracellular Ca2؉ -dependent cysteine proteases that are released in the extracellular milieu by tubular epithelial cells following renal ischemia. Here we show that externalized calpains increase epithelial cell mobility and thus are critical for tubule repair. In vitro, exposure of human tubular epithelial cells (HK-2 cells) to -calpain limited their adhesion to extracellular matrix and increased their mobility. Calpains acted primarily by promoting the cleavage of fibronectin, thus preventing fibronectin binding to the integrin ␣ v ␤ 3 . Analyzing downstream integrin effects, we found that the cyclic AMP-dependent protein kinase A pathway was activated in response to ␣ v ␤ 3 disengagement and was essential for calpain-mediated increase in HK-2 cell mobility. In a murine model of ischemic acute renal failure, injection of a fragment of calpastatin, which specifically blocked calpain activity in extracellular milieu, markedly delayed tubule repair, increasing functional and histological lesions after 24 and 48 h of reperfusion. These findings suggest that externalized calpains are critical for tubule repair process in acute renal failure.Calpains are intracellular Ca 2ϩ -dependent cysteine proteases (1). The major isozymes, calpain 1 or -calpain and calpain 2 or m-calpain, are distributed ubiquitously and activated in vitro by micromolar and millimolar concentrations of Ca 2ϩ , respectively. They are heterodimers composed of a ϳ80-kDa catalytic subunit (encoded by CAPN1 and CAPN2 for -and m-calpain, respectively) and a common ϳ30-kDa regulatory subunit (encoded by CAPN4). Binding of Ca 2ϩ to -or m-calpain induces the release of constraints imposed by domain interactions and results in a two-stage activation process: first, the release of ϳ30-kDa regulatory subunit; and second, the rearrangement of the active site cleft in ϳ80-kDa catalytic subunit (2). Calpain activity is tightly controlled by calpastatin, a specific endogenous inhibitor, which contains four equivalent inhibitory domains (1). By conducting limited proteolysis of intracellular substrates, calpain activity has been shown to be critical for a great diversity of cellular responses. They include rearrangement of cytoskeletal linkages to the plasma membrane during cell adhesion and mobility, modification of molecules in signal transduction pathways, degradation of enzymes controlling the cell cycle, and activation of proteolytic cascades leading to cell apoptosis or necrosis (1,3,4).Recently, several groups showed that calpains may be released from cells into the extracellular environment and thus may have an extracellular role. Like other intracellular enzymes, calpains may indeed leak out from injured and dying cells such as hepatocytes exposed to toxic chemicals (5). The release of intracellular calpains from blood mononuclear cells (6), osteoblasts (7), chondrocytes (8), and parathyroid cells (9) is not due to cell death, but rather to a nonclassical pathway of secretion, which involves in certain cells the shedding of membrane vesi...

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Section: Discussionmentioning

confidence: 97%

Section: Extracellular -Calpain Modulates the Behavior Of Proximalmentioning

confidence: 99%

Extracellular Calpains Increase Tubular Epithelial Cell Mobility

Frangié¹,

Zhang

Perez

et al. 2006

Journal of Biological Chemistry

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“…Pc-1 has been found to be tyrosine-phosphorylated and phosphorylated by Protein kinase A (13,14). Protein kinase X, a novel cAMP-dependent serine/ threonine kinase that regulates epithelial migration and morphogenesis is aberrantly expressed in kidney tissues from ADPKD patients (15). Mutations in Nek-family kinases Nek1 and Nek8 underlie polycystic kidney phenotypes in a pair of naturally occurring mouse models (16,17).…”

mentioning

confidence: 99%

Calcium Dependence of Polycystin-2 Channel Activity Is Modulated by Phosphorylation at Ser812

Cai

Anyatonwu

Okuhara

et al. 2004

Journal of Biological Chemistry

139

188

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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 found that this constitutive phosphorylation occurs at Ser 812 , a putative casein kinase II (CK2) substrate domain. Ser 812 can be phosphorylated by CK2 in vitro and substitution S812A results in failure to incorporate phosphate in cultured epithelial cells. Non-phosphorylated forms of PC-2 traffic normally in the endoplasmic reticulum and cilial compartments and retain homo-and hetero-multimerization interactions with PC-2 and polycystin-1, respectively. Single-channel studies of PC-2, S812A, and a substitution mutant, T721A, not related to phosphorylation show that PC-2 and S812A function as divalent cation channels with similar current amplitudes across a range of holding potentials; the T721A channel is not functional. Channel open probabilities for PC-2 and S812A show a bell-shaped dependence on cytoplasmic Ca 2؉ but there is a shift in this Ca 2؉ dependence such that S812A is 10-fold less sensitive to Ca 2؉ activation/inactivation than the wild type PC-2 channel. In vivo analysis of PC-2-dependent enhanced intracellular Ca 2؉ transients found that S812A resulted in enhanced transient duration and relative amplitude intermediate between control cells and those overexpressing wild type PC-2. Phosphorylation at Ser 812 modulates PC-2 channel activity and factors regulating this phosphorylation are likely to play a role in the pathogenesis of polycystic kidney disease.

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“…Finally, some bona fide interactors may have not been present in the prey library due either to construction artifacts or to weak or absent expression in the mostly mature population of cells from which mRNA for the library was constructed. It is becoming apparent that the PrKX protein is expressed in a number of other tissue sources (Blaschke et al, 2000;Li et al, 2002), so interactions that occur with proteins specific to these tissues will be missed as well. Those caveats aside, the recovered interactors appear to lend credence to the quality of the screen.…”

Section: Discussionmentioning

confidence: 99%

“…A second interactor of PrKX was identified by a yeast two-hybrid approach as the Rep78 protein of adeno-associated virus (AAV; 12), whose interaction with PrKX reportedly modulates viral interference between AAV and adenovirus (Di Pasquale and Chiorini, 2003). More recently, PrKX was found to also play a role in neuronal differentiation (Blaschke et al, 2000) and in renal cell migration and morphogenesis (Li et al, 2002). However, to this date no innate cell substrates for the phosphorylation activity of this kinase during cell maturation have been identified.…”

Section: Introductionmentioning

confidence: 99%

Smad6 is a protein kinase X phosphorylation substrate and is required for HL-60 cell differentiation

Glesne

Huberman

2006

Oncogene

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To gain insight into the function of human protein kinase X (PrKX), a signal-transduction protein required for macrophage differentiation, we identified regulatory subunit I alpha of protein kinase A, T54 and Smad6 as partners for this protein using a yeast two-hybrid interaction screen. Interactions between PrKX and these proteins were substantiated by co-immunoprecipitation. Interaction between Smad6 and PrKX was also confirmed in human myeloid HL-60 cells following their phorbol 12-myristate 13-acetate (PMA)-induced differentiation into macrophages. In vitro phosphorylation assays demonstrated that PrKX phosphorylates Smad6 at a serine residue. Mutagenesis of this site resulted in abrogation of PrKX phosphorylation. Both PrKX and Smad6 were shown to be co-localized to the nuclear compartment of HL-60 cells during their macrophage differentiation where PrKX levels are induced and Smad6 protein levels remain relatively constant while levels of serine phosphorylation of Smad6 increase. By using in vitro electrophoretic mobility shift assays and in vivo chromatin immunoprecipitation, we also demonstrate that during macrophage differentiation Smad6 displays an increased binding to the human osteopontin, Id2, and Hex gene promoters, which correlates to an observed increased expression of these genes. Finally, vector-based RNA interference experiments established that both Smad6 and PrKX proteins are required for PMA-induced cell attachment and spreading.

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PRKX, a phylogenetically and functionally distinct cAMP-dependent protein kinase, activates renal epithelial cell migration and morphogenesis

Cited by 48 publications

References 48 publications

Extracellular Calpains Increase Tubular Epithelial Cell Mobility

Extracellular Calpains Increase Tubular Epithelial Cell Mobility

Calcium Dependence of Polycystin-2 Channel Activity Is Modulated by Phosphorylation at Ser812

Smad6 is a protein kinase X phosphorylation substrate and is required for HL-60 cell differentiation

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