Phosphorylation of HSP27 by Protein Kinase D Is Essential for Mediating Neuroprotection against Ischemic Neuronal Injury

Abstract

Heat shock protein 27 (HSP27, or HSPB1) exerts cytoprotection against many cellular insults, including cerebral ischemia. We previously identified apoptosis signal-regulating kinase 1 (ASK1) as a critical downstream target of HSP27 conferring the neuroprotective effects of HSP27 against neuronal ischemia. However, the function of HSP27 is highly influenced by post-translational modification, with differential cellular effects based on phosphorylation at specific serine residues. The role of phosphorylation in neuronal ischemic neuroprotection is currently unknown. We have created transgenic mice and viral vectors containing HSP27 mutated at three critical serine residues (Ser15, Ser78 and Ser82) to either alanine (HSP27-A, non-phosphorylatable) or aspartate (HSP27-D, phospho-mimetic) residues. Under both in vitro and in vivo neuronal ischemic settings, overexpression of wild-type HSP27 (HSP27) and HSP27-D, but not HSP27-A, was neuroprotective and inhibited downstream ASK1 signaling pathways. Consistently, overexpressed HSP27 was phosphorylated by endogenous mechanisms when neurons were under ischemic stress, and single point mutations identified Ser15 and Ser82 as critical for neuroprotection. Using a panel of inhibitors and gene knockdown approaches, we identified the upstream kinase protein kinase D (PKD) as the primary kinase targeting HSP27 directly for phosphorylation. PKD and HSP27 co-immunoprecipitated, and inhibition or knockdown of PKD abrogated the neuroprotective effects of HSP27 as well as the interaction with and inhibition of ASK1 signaling. Taken together, these data demonstrate that HSP27 requires PKD-mediated phosphorylation for its suppression of ASK1 cell death signaling and neuroprotection against ischemic injury.

Endothelium-targeted overexpression of heat shock protein 27 ameliorates blood–brain barrier disruption after ischemic brain injury

“…Under physiological conditions, the level of HSP27 is extremely low in the brain; however, HSP27 expression is transiently induced in brain glial cells and/or ECs at 24 h or later following cerebral I/R (14). Previous studies by our and other groups demonstrated robust neuroprotective effects of HSP27 overexpression against I/R brain injury (15)(16)(17)(18), which were largely credited to its antiapoptotic actions. However, HSP27 exerts additional actions on ECs (14).…”

“…S4). Moreover, the expression of endogenous HSP27 is induced in postischemic brain mainly in nonneuronal cells, glia (18) as well as ECs (22), raising the possibility that HSP27 works through additional mechanisms. 3 .…”

“…We previously reported that transgenic mice with ubiquitous overexpression of HSP27 developed significantly less brain damage after cerebral I/R, including less BBB disruption (17,18,22). These beneficial effects of HSP27 were attributed mainly to its well-characterized antiapoptotic functions Fig.…”

“…At 24 h after tFCI, the area with loss of microtubule-associated protein 2 (MAP2) immunofluorescence illustrates the infarct zone on adjacent sections from the same brains. in neurons (17,18) and ECs (22). However, neuronal, glial, or endothelial HSP27 may have distinct effects on the pathogenesis of I/R brain injury, which would not be distinguishable in global HSP27-overexpressing mice.…”

Protein kinase D: a new player among the signaling proteins that regulate functions in the nervous system

“…PKD also directly phosphorylates HSP27 [66] . PKD loss-of-function abolishes the neuroprotective effects of HSP27 [67] . …”