Fig 2: Relationship between phosphorylation and subcellular localization of HSPB5. (a) Localization of overexpressed recombinant HSPB5 and endogenous PLK2. After 5 µM MG132 treatment (24 h), FLAG-tagged HSPB5 and endogenous PLK2 in undifferentiated L6 cells were immunofluorescently stained with anti-tag and anti-PLK2 antibodies. (b) Effect of PLK2 on localization of HSPB5 and desmin protein. Endogenous HSPB5 and desmin were immunofluorescently stained with anti-HSPB5 and anti-desmin antibodies. Undifferentiated L6 cells were transfected with siRNA to knockdown PLK2. After differentiation (48 h), L6 myotubes were treated with MG132 for 24 h. White arrowheads indicate cells with colocalization of HSPB5 and desmin at perinuclear ER region. Scale bar indicates 20 µm.

Fig 3: PLK2 phosphorylates HSPB5 at serine 19 under ER stress. (a) Knockdown efficiency of PLK2 by siRNA. L6 cells were transfected with siRNA to knockdown PLK2 and treated with 5 µM MG132 for 24 h. Graph presents relative value of expression level of PLK2. Data in graphs represent means ± standard error (n = 3). Statistical analyses were performed using t-test. ** p < 0.01. (b) Schematic representation of three known phosphorylation sites of HSPB5. Arrows indicate name of the kinase catalyzing phosphorylation. (c) Effect of PLK2 on each phosphorylation site. Undifferentiated L6 cells were transfected with siRNA to knockdown PLK2. After differentiation (48 h), L6 myotubes were treated with 5 µM MG132 for 24 h. Phosphorylation level was detected by Western blotting with specific antibodies. Graph presents ratio of phosphorylated HSPB5/HSPB5 after quantification of each band. Data in graphs represent means ± standard error (n = 3). Statistical analyses were performed using one-way ANOVA with Tukey test. ‡ p < 0.01, † p < 0.05; n.s. not significant. (d) Effect of PLK2 activity on phosphorylation of serine 19. After differentiation (48 h), L6 myotubes were treated with 10 nM BI2536 and 5 µM MG132 for 24 h. Phosphorylation level was detected by Western blotting with specific antibodies.

Fig 4: A novel PLK2/HSPB5 pathway is proposed as a model for cytoprotection against ER stress by MG132. PLK2, a novel binding protein of HSPB5, phosphorylates serine 19 (pSer19) of HSPB5 under ER stress conditions caused by proteome inhibitor MG132. This phosphorylation is responsible for inhibitory effect of HSPB5 on ER stress-induced apoptosis by MG132. PLK2/HSPB5 pathway provides a molecular mechanism for pSer19 of HSPB5 and represents part of mechanism for suppressing ER stress-induced apoptosis via proteasome inhibition.

Fig 5: Construction of HSPB5-BioID2 expression plasmid and BioID activity. (a) Schematic diagram of BioID2 fusion protein. (b) Subcellular localization of HSPB5-BioID2 expression. HeLa cells were transfected with expression plasmid, and 24 h after addition of biotin (50 µM), cells were immunostained with anti-HA antibody and stained with fluorescently labeled secondary antibody, streptavidin-Alexa Fluor 546, and with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI). White arrows indicate lamellipodia around cell membrane. Scale bar indicates 20 µm. (c) Detection of BioID2 fusion protein expression and BioID activity. 293T cells were transfected with expression plasmid, and cells were collected 24 h after addition of biotin. Expression of BioID2 fusion protein was detected with an anti-HA antibody. (d) Biotinylated proteins were detected using streptavidin-peroxidase polymer. Arrowheads (HSPB1-BioID2) and arrows (HSPB5-BioID2) indicate positions of respective proteins. HSPB1 is an isoform that belongs to the same sHsp family as HSPB5.