Serine/threonine phosphatase 5 (PP5C/PPP5C) regulates the ISOC channel through a PP5C‐FKBP51 axis

Abstract

Pulmonary endothelial cells express a store-operated calcium entry current ( I), which contributes to inter-endothelial cell gap formation. I is regulated by a heterocomplex of proteins that includes the immunophilin FKBP51. FKBP51 inhibits I by mechanisms that are not fully understood. In pulmonary artery endothelial cells (PAECs) we have shown that FKBP51 increases microtubule polymerization, an event that is critical for I inhibition by FKBP51. In neurons, FKBP51 promotes microtubule stability through facilitation of tau dephosphorylation. However, FKBP51 does not possess phosphatase activity. Protein phosphatase 5 (PP5C/PPP5C) can dephosphorylate tau, and similar to FKBP51, PP5C possesses tetratricopeptide repeats (TPR) that mediate interaction with heat shock protein-90 (HSP90) chaperone/scaffolding complexes. We therefore tested whether PP5C contributes to FKBP51-mediated inhibition of I. Both siRNA-mediated suppression of PP5C expression in PAECs and genetic disruption of PP5C in HEK293 cells attenuate FKBP51-mediated inhibition of I. Reintroduction of catalytically competent, but not catalytically inactive PP5C, restored FKBP51-mediated inhibition of I. PAEC cell fractionation studies identified both PP5C and the ISOC heterocomplex in the same membrane fractions. Further, PP5C co-precipitates with TRPC4, an essential subunit of ISOC channel. Finally, to determine if PP5C is required for FKBP51-mediated inhibition of calcium entry-induced inter-endothelial cell gap formation, we measured gap area by wide-field microscopy and performed biotin gap quantification assay and electric cell-substrate impedance sensing (ECIS®). Collectively, the data presented indicate that suppression of PP5C expression negates the protective effect of FKBP51. These observations identify PP5C as a novel member of the ISOC heterocomplex that is required for FKBP51-mediated inhibition of I.

S100A6 is a positive regulator of PPP5C‐FKBP51‐dependent regulation of endothelial calcium signaling

“…We recognize however that upon I SOC activation with rolipram/thapsigargin it is likely that other thapsigargin‐activated calcium channels open as well, and using the Fura2 approach, we are not be able to discriminate between different sources of calcium entry. Thus, to specifically determine the role of S100A6 in ISOC channel activation, we next performed patch‐clamp electrophysiology using the whole‐cell, voltage‐clamp configuration as described previously . We measured I SOC in the doxycycline‐inducible S100A6 over‐expressing cells in the presence or absence of doxycycline.…”

“…In PAECs, treatment with the calcium ATPase inhibitor thapsigargin (1.0 µM) activates I SOC . [1][2][3][4][5][6]8,9 However, in PMVECs treatment with thapsigargin alone is insufficient to activate I SOC . 8 To activate I SOC in PMVECs, we first incubated the cells with the type IV phosphodiesterase inhibitor rolipram (0.1 µM) for 15 minutes followed by treatment with thapsigargin (1.0 µM).…”

“…The TRPC4 subunit in particular is required for channel activity through its interaction with the spectrin membrane skeleton . In addition to interacting with the spectrin membrane skeleton, the channel interacts with a number of accessory proteins, overall comprising what we have termed the ISOC heterocomplex . For instance, we have previously shown that Orai1 associates with and modulates ISOC channel activity .…”

“…For instance, we have previously shown that Orai1 associates with and modulates ISOC channel activity . We have also shown that the FK506‐binding protein 51 (FKBP51), interacts with TRPC4 and inhibits I SOC . The serine/threonine protein phosphatase 5 (PPP5C; encoded by PPP5C ), is also part of the heterocomplex and is needed for FKBP51‐mediated inhibition of I SOC .…”

“…Transfection of siRNA to PMVECs was achieved as previously described. 4,6 PMVEC monolayers were grown to 50%-60% confluency. Cell monolayers were then transfected with a 20 nM final siRNA concentration in lipofectamine 3000 (Thermo Fisher Scientific).…”