Wang et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-023-00481-6(2023) 24:19

BMC Molecular and Cell
BiologyOpen Access

RESEARCH

SUMOylation of PDGF receptor α affects
signaling via PLCγ and STAT3, and cellproliferation
Kehuan Wang1, Natalia Papadopoulos1, Anahita Hamidi1, Johan Lennartsson2 and Carl-Henrik Heldin1*
Abstract
Background The platelet-derived growth factor (PDGF) family of ligands exerts their cellular effects by binding to
α- and β-tyrosine kinase receptors (PDGFRα and PDGFRβ, respectively). SUMOylation is an important posttranslational
modification (PTM) which regulates protein stability, localization, activation and protein interactions. A mass
spectrometry screen has demonstrated SUMOylation of PDGFRα. However, the functional role of SUMOylation of
PDGFRα has remained unknown.
Results In the present study, we validated that PDGFRα is SUMOylated on lysine residue 917 as was previously
reported using a mass spectrometry approach. Mutation of lysine residue 917 to arginine (K917R) in PDGFRαsubstantially decreased SUMOylation, indicating that this amino acid residue is a major SUMOylation site. Whereas
no difference in the stability of wild-type and mutant receptor was observed, the K917R mutant PDGFRα was lessubiquitinated than wild-type PDGFRα. The internalization and trafficking of the receptor to early and late endosomes
were not affected by the mutation, neither was the localization of the PDGFRα to Golgi. However, the K917R mutant
PDGFRα showed delayed activation of PLC-γ and enhanced activation of STAT3. Functional assays showed that the
mutation of K917 of PDGFRα decreased cell proliferation in response to PDGF-BB stimulation.
Conclusions SUMOylation of PDGFRα decreases ubiquitination of the receptor and affects ligand-induced signaling
and cell proliferation.
Keywords PDGFRα, Receptor tyrosine kinase, SUMO, SUMOylation*Correspondence:
Carl-Henrik Heldinc-h.heldin@imbim.uu.se
1
Department of Medical Biochemistry and Microbiology, Uppsala
University, UppsalaBox 582, Sweden
2
Department of Pharmaceutical Biosciences, Uppsala University, Uppsala,SwedenIntroduction
Signaling by members of the platelet-derived growth
factors (PDGF) family, consisting of dimeric isoformsof A-, B-, C- and D-polypeptide chains, has been implicated in embryonal development, wound healing, andtissue homeostasis in the adult, as well as in cancer [1].
PDGF isoforms bind to α- and β-tyrosine kinase receptors (PDGFRα and PDGFRβ, respectively), promoting
dimerization of receptors, which in turn causes receptor autophosphorylation, followed by ubiquitinationand internalization from the cell membrane. PDGFAA specifically induces α-receptor homodimers, while© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Wang et al. BMC Molecular and Cell Biology(2023) 24:19

PDGF-BB induces both α- and β-receptor homodimers,
as well as an α/β receptor heterodimer. Activation of
PDGF receptors initiates downstream signaling pathways
promoting cell migration and proliferation.
The activity, stability and subcellular localization ofproteins are controlled by different posttranslational
modifications (PTMs), including phosphorylation on
serine/threonine or tyrosine residues, acetylation, methylation, ubiquitination, SUMOylation, and others [2,3]. Ubiquitination of proteins can occur by addition of
single ubiquitin molecules (mono-ubiquitination), or
by addition of poly-ubiquitin chains linked via different
lysine residues in ubiquitin [4]. In addition, proteins canbe modified by certain ubiquitin-like molecules, including five SUMO isoforms, i.e., SUMO1, 2, 3, 4 or 5 [5].
SUMO1 and SUMO2/3 are ubiquitously expressed andconjugated to target proteins differently. Thus, SUMO2/3
can form poly-SUMOylation chains, while SUMO1 takes
part in mono-SUMOylation of proteins and is able to terminate SUMO2/3 polymers [6, 7]. Several receptor tyrosine kinases have been reported to be the substrates of
the SUMOylation machinery [8–10]. Both ubiquitinationand SUMOylation play important roles in the regulationof protein stability, localization, activation and interaction; however, the conjugation of ubiquitin and SUMO tothe same protein may lead to different consequences. For
example, ubiquitination often induces protein degradation, while SUMO may stabilize its target proteins [11].
PDGFRα and PDGFRβ activate similar signaling pathways, but certain differences have been reported [12, 13].
Both receptors are able to activate ERK1/2 MAP-kinase,albeit with different kinetics and efficiency [14], as well as
phosphatidylinositol 3´-kinase (PI3K), Src and phospholipase C-γ (PLC-γ) pathways [15]. It has been reported
that PDGFRα is stored inactive in the cytoplasm, likely
in Golgi-derived vesicles, while trafficking to cilia is necessary for controlled activation of PDGFRα signaling[16]. In contrast, PDGFRβ is able to signal continuously
from the plasma membrane [17] and the intact PDGFRβis also translocated to the nucleus where it affects chromatin remodeling [18]. The activation, internalization,
stability and signaling properties of PDGFRs has beenshown to be regulated by phosphorylation and ubiquitination [1, 19]. No SUMOylation has been reported
for PDGFRβ, whereas PDGFRα has been demonstrated
to be SUMOylated in a mass spectrometry screen [20,21]. The purpose of this work was to explore the role of
SUMOylation in the signaling, stability and subcellular
localization of PDGFRα.

Results
PDGFRα is SUMOylated

In order to validate the finding by a mass spectrometry
analysis that PDGFRα is SUMOylated [20], we transiently

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co-overexpressed PDGFRα and HA-SUMO1 in COS-7
cells and subjected cell lysates to immunoprecipitation
with an antibody against PDGFRα, followed by immunoprecipitation with an HA-antibody; SUMOylation of
PDGFRα was detected under starvation conditions andpeaked at 45 min of stimulation with 20 ng/ml PDGFAA (Fig. 1a; quantified in 1b). In PAE cells stably transfected with PDGFRα, the receptor was also found to be
SUMOylated, as determined by immunoprecipitation ofendogenous SUMO1 and immunoblotting for PDGFRα(Fig. 1c). We then performed co-immunoprecipitation experiments using RPE1 cells that express endogenous PDGFRα and SUMO1. We found that PDGF-AA
induced the SUMOylation of PDGFRα, and that more
SUMOylation was observed in cell cultures that were not
starved (Fig. 1d). To investigate whether the observed
SUMOylation was on PDGFRα, or on some other proteins bound to PDGFRα, we boiled the lysates before
immunoprecipitation to remove all PDGFR-boundproteins; SUMOylation of PDGFRα was detected also
after this treatment, while no SUMO1 modification of
PDGFRβ was seen (Fig. 1e). The amount of SUMOylated
PDGFRα under denaturing conditions was decreased
after 45 min of PDGF stimulation, suggesting that theobserved SUMOylation of PDGFRα, as shown in Fig. 1A,
may include not only SUMOylated PDGFRα, but also
other PDGFRα-bound SUMOylated proteins. The addition of ginkgolic acid (GA), an E1 activating enzyme
inhibitor, impaired the SUMOylation of PDGFRα or
associated proteins (Fig. 1f ).
Mutation of K917 abolishes SUMOylation of PDGFRαLysine residue 917 is the only reported SUMOylationsite in PDGFRα so far [20]. We mutated this residue toan arginine residue (K917R) to investigate if this is indeed
an acceptor site for SUMO1. HEK293T cells were transiently co-transfected with wild-type (WT) or K917R
mutant PDGFRα and HA-SUMO1. After stimulation
with 20 ng/ml PDGF-AA for 45 min under starvation
conditions, SUMOylation of WT PDGFRα was determined by immunoprecipitation of PDGFRα, followed
by immunoblotting with an HA antibody; significantly
less SUMOylation was detected on the K917R mutant ascompared to the WT PDGFRα (Fig. 2a; quantified in 2b).
These results suggest that K917 is a main SUMOylationsite in PDGFRα, but we cannot exclude that other acceptor sites exist since there was still some SUMOylation left
on the mutated receptor.
SUMOylation at K917 does not affect the stability of
PDGFRαIt has been reported that protein SUMOylation may
enhance the stability of its substrates by inhibiting its
ubiquitination [22, 23]. In order to investigate the effectWang et al. BMC Molecular and Cell Biology(2023) 24:19

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Fig. 1 PDGFRα is SUMOylated. (a) COS-7 cells were co-transfected with indicated plasmids, followed by serum-starvation for 24 h. Cells were stimulatedwith 20 ng/ml PDGF-AA or PDGF-BB for different time periods, lysed and subjected to immunoprecipitation using an antibody against PDGFRα, followed
by immunoblotting using anti-HA and anti-PDGFRα antibodies. Non-immune IgG was used as a negative control. Total cell lysates (TCL) were immunoblotted with an anti-PDGFRα antibody. HP95 (Alix) was used as a loading control. (b) Quantification of SUMOylated PDGFRα in panel A after different timesof stimulation of PDGF-AA. Four independent experiments were performed and the standard deviation is indicated. (c) PAE cells with stably transfected
PDGFRα were stimulated with 20 ng/ml PDGF-AA for 0, 1 and 4 h after serum-starvation for 24 h. Immunoprecipitation of SUMO1 was performed and
SUMOylation of PDGFRα was determined by immunoblotting with a PDGFRα antibody. (d) RPE1 cells were stimulated with 20 ng/ml PDGF-AA for 0 and
45 min after serum-starvation or the presence of 10% FBS for 24 h. After immunoprecipitation with a SUMO1 antibody, samples were subjected to immunoblotting with a PDGFRα antibody. Receptor expression and loading control (Alix) were determined by immunoblotting of total cell lysates. (e) RPE1
cells, starved or not, were treated as in panel d, and cell lysates were heated at 95℃ and sonicated before immunoprecipitation with a SUMO1 antibody,followed by immunoblotting with antibodies against PDGFRα and PDGFRβ. (f) COS-7 cells transfected with PDGFRα and HA-SUMO1 were serum-starved
and treated with 20 µM ginkgolic acid for 24 h before stimulation with PDGF-AA. SUMO1 was precipitated with HA antibody and PDGFRα was determinedby immunoblotting. All experiments were performed three times or more except for the experiments shown in panels c and f which were performed twotimes. The immunoblots were cropped for clarity. Full length blots are presented in Figure S1of SUMOylation on PDGFRα stability, WT or K917R
mutant PDGFRα were overexpressed in COS-7 cells. The
transfected cells were treated with cycloheximide (CHX)
to inhibit the synthesis of new proteins, and after different time periods up to 6 h, cell lysates were preparedand subjected to immunoblotting for PDGFRα (Fig. 3a;quantified in 3b). No significant difference in the steadystate levels of WT and K917R mutant PDGFRα overtime was observed. The stability of PDGFRα in response
to PDGF-AA stimulation was then investigated in PAE
cells transiently transfected with WT or K917R mutant

PDGFRα. Upon PDGF-AA stimulation, the levels of WT
and K917R mutant PDGFRα were similar (Fig. 3c; quantified in 3d). In order to make sure that these results were
not due to low efficiency of transfection, tet-inducible
PAE cell lines were established in which the expressionof WT or K917R mutant PDGFRα could be induced bytreatment of cells with doxycycline. After induction of
PDGFRα expression for 48 h, there was no significant
difference of PDGF-AA-induced degradation of WT andmutant PDGFRα (Fig. 3e; quantified in 3f), which is consistent with the result using transiently transfected PAE

Wang et al. BMC Molecular and Cell Biology(2023) 24:19

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Fig. 2 Mutation of lysine residue 917 impairs SUMOylation of PDGFRα. (a) HEK293T cells were transfected with WT or K917R mutant PDGFRα and HASUMO1. After serum-starvation for 24 h, cells were stimulated with PDGF-AA for 45 min, followed by immunoprecipitation with an anti-PDGFRα antibody.
The proteins then were separated by SDS-PAGE and subjected to immunoblotting with indicated antibodies. The expression of PDGFRα was determinedby immunoblotting of total cell lysates (TCL). The immunoblots were cropped for clarity. Full length blots are presented in Figure S1. (b) SUMOylated
PDGFRα was quantified. SUMOylation levels of WT PDGFRα with stimulation of PDGF-AA at 0 min were set as 1. The experiment was performed threetimes and a representative blot is shown and quantified

cells. PDGF receptors are degraded in lysosomes [24]and proteasomes [19, 25]. Incubation with the lysosomal
inhibitor chloroquine (CQ) inhibited the degradationof both WT and K917R mutant PDGFRα to the same
extent; the proteasomal inhibitor bortezomib (BTZ) also
inhibited the degradation of both WT and K917R mutant
PDGFRα slightly, but this was not significant (Fig. 3g;
quantified in 3 h). We observed that the expression levels of the K917R mutant were sometimes lower than thelevels of WT PDGFRα, which could be restored withaddition of chloroquine, thus suggesting that a fraction of
mutated receptor may be incorrectly folded and targeted
for lysosomal degradation.
Mutation of K917 attenuates ubiquitination of PDGFRα

We next investigated the impact of SUMOylation of K917
on the ubiquitination of PDGFRα. PAE cells transiently
transfected with HA-tagged WT or K917R mutant
PDGFRα were subjected to immunoprecipitation witha PDGFRα antibody, followed by immunoblotting withan antibody against ubiquitin. A decrease of ubiquitination of the K917R mutant PDGFRα, compared to the WT
receptor, was observed in response to PDGF stimulation(Fig. 4a). Similarly, when WT or K917R mutant PDGFRα
was induced in tet-inducible PAE cells, the K917R mutant
was less ubiquitinated relative to WT PDGFRα (Fig. 4b).
Mutation of K917 does not affect the internalization of
PDGFRαOur results indicated that the K917R mutant PDGFRαis less ubiquitinated than WT PDGFRα. Ubiquitinationof PDGFRs is linked to internalization of the receptors[19, 26]. SUMOylation has also been reported to affectinternalization of its target proteins [27, 28]. In order toinvestigate whether SUMOylation of PDGFRα affects
its internalization, we determined the level of WT and
K917R mutant PDGFRα on the cell surface by biotinylating the cell surface receptors after different time periodsof stimulation with PDGF-AA. We did not observe any
difference in internalization of the K917R mutant
PDGFRα as compared to the WT receptor in transfected
PAE cells (Fig. 5a; quantified in 5b). Similarly, the internalization of PDGFRα stably expressed in tet-inducible
cell lines was not affected by the K917R mutation (Fig. 5c;quantified in 5d).
Mutation of K917 does not affect the localization of
PDGFRα to the Golgi apparatus and endosomes

SUMOylation of certain RTKs has been shown to affecttheir subcellular localization [8–10]. In order to investigate whether SUMOylation affects the subcellular localization of PDGFRα, we first determined the localizationof WT and K917R mutant PDGFRα to the Golgi in
response to PDGF-AA stimulation. Immunofluorescentlabeling of PDGFRα and GM130, a marker of Golgi, demonstrated similar localization of WT and K917R mutant
PDGFRα at the Golgi apparatus (Fig. 6a). The traffic of
WT and K917R mutant PDGFRα to early and late endosomes, in response to stimulation with PDGF-BB, wasalso investigated by co-staining of PDGFRα with an earlyendosome marker EEA1 and a late endosome marker
Rab7. The results revealed similar localization and WT
or K917R mutant PDGFRα to early and late endosomes(Fig. 6b and c).
Mutation of K917 affects the activation of PLC-γ and STAT3

We then investigated the impact of the mutation of
K917 on the downstream signaling pathways induced by
PDGFRα activation, using immunoblotting with phospho-specific antibodies. By transiently transfecting PAE
cells with WT or K917R mutant PDGFRα, and stimulating the cells with PDGF-AA for different time periods(Fig. 7a), we observed that the phosphorylation of PLC-γ
was delayed in cells expressing K917R mutant PDGFRα,peaking at 45 min of stimulation of PDGF-AA (Fig. 7b),and that phosphorylation of STAT3 was enhanced

Wang et al. BMC Molecular and Cell Biology(2023) 24:19

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Fig. 3 Mutation of lysine residue 917 does not affect the steady state level or ligand-induced degradation of PDGFRα. (a) WT or K917R mutant PDGFRαwere transfected in COS-7 cells, followed by treatment with 50 µg/ml cycloheximide for 0, 1, 2, 4 and 6 h. The cells were then lysed and immunoblottedwith antibodies against PDGFRα and Alix as a loading control. (b) The expression of PDGFRα was quantified. WT or K917R mutant PDGFRα levels at 0 h
treatment of cycloheximide was set at 1. (c-f) WT or K917R mutant PDGFRα were transiently transfected into PAE cells (c) or induced with doxycycline in
PAE-PDGFRα tet-inducible cell line (e), followed by serum-starvation for 24 h and treatment with 50 µg/ml cycloheximide for 1 h. Cells were then stimulated with PDGF-BB for 0, 0.5, 1 or 2 h, after which the amount of PDGFRα was examined by immunoblotting, and quantified relative to the levels of Alix or
α-tubulin which served as loading controls (d, f). The results of three independent experiments were quantified. Expression levels of WT or K917R mutant
PDGFRα without PDGF-AA stimulation were set at 1. (g) Tet-inducible PAE cells were treated with doxycycline for 48 h to induce the expression of WT or
K917R mutant PDGFRα and starved, followed by incubation with 25 µM chloroquine (CQ) or 1 µM bortezomib (BTZ) for 4 h; DMSO served as a control.
Cells were then stimulated with 20 ng/ml PDGF-AA for 45 min and lysed for immunoblotting. (h) Quantification of the results of panel g. The level of WTPDGFRα without inhibitor treatment and PDGF-AA stimulation was set as 1. All experiments were performed three times or more except for panels a andg which were performed two times. The immunoblots were cropped for clarity. Full length blots are presented in Figure S1

Wang et al. BMC Molecular and Cell Biology(2023) 24:19

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Fig. 4 Mutation of lysine residue 917 decreases the ubiquitination of PDGFRα after short-term ligand stimulation. PAE cells transfected with either HAtagged WT or K917R mutant PDGFRα (a), or tet-inducible PAE-PDGFRα cells induced with doxycycline to overexpress WT or K917R mutant PDGFRα (b),were serum-starved for 24 h and then stimulated with PDGF-AA for indicated time periods. An antibody against HA or PDGFRα was used to immunoprecipitate PDGFRα, followed by immunoblotting with indicated antibodies. Total cell lysates were also subjected to immunoblotting with antibodies
against HA, PDGFRα, and α-tubulin which was used as a loading control. Ub, ubiquitin. Each experiment was performed three times or more. The immunoblots were cropped for clarity. Full length blots are presented in Figure S1

Fig. 5 Internalization of PDGFRα from the cell surface is not affected by mutation of lysine residue 917. (a) PAE cells were transfected with WT or K917R
mutant PDGFRα and serum-starved for 24 h, followed by stimulation with 20 ng/ml PDGF-AA for indicated time periods. Cell surface proteins werethen biotinylated. After lysis of cells, biotinylated proteins were collected on streptavidin-agarose beads. Adsorbed proteins and total cell lysates were
subjected to SDS-PAGE and immunoblotted with antibodies against PDGFRα, transferrin receptor (TfR) or Alix. Alix was used as a loading control. (b) The
expression level of PDGFRα remaining on the cell surface compared with transferrin receptor (TfR) from three independent repeats was quantified. The
ratio at 0 min of PDGF-AA stimulation was set at 1. (c) Expression of WT or K917R mutant PDGFRα in a tet-inducible PAE cell line was induced with doxycycline and assessed as described in panel a. (d) The PDGFRα levels on the cell surface relative to TfR on the cell surface from three independent repeats wasquantified. The ratio of induced and unstimulated cells was set as 1. The immunoblots were cropped for clarity. Full length blots are presented in Figure S1

Wang et al. BMC Molecular and Cell Biology(2023) 24:19

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Fig. 6 Mutation of lysine residue 917 does not affect the subcellular localization of PDGFRα. (a) Tet-inducible PAE cells were grown without doxycycline(-Dox) or treated with doxycycline (+ Dox) to induce the expression of WT or K917R mutant PDGFRα, and then stimulated, or not, with 20 ng/ml PDGF-AA
for 45 min, fixed and stained with DAPI and primary antibodies against PDGFRα and the Golgi marker GM130, followed by fluorescent secondary antibodies. (b, c) Tet-inducible PAE cells were induced to express WT or K917R mutant PDGFRα, followed by staining with DAPI and with antibodies against
PDGFRα, and markers for early (EEA1; b) and late (Rab7; c) endosomes

Wang et al. BMC Molecular and Cell Biology(2023) 24:19

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Fig. 7 Mutation of lysine 917 affects PDGFRα activation of PLCγ and STAT3 and cell proliferation. (a) PAE cells were transiently transfected with WT or
K917R mutant PDGFRα, serum-starved and stimulated with 20 ng/ml PDGF-AA for indicated time periods. Expression levels of phosphorylated PLCγ (b),STAT3 (c), Akt (d) and ERK1/2 (e) and total proteins were determined using antibodies against phosphorylated PDGFRα (pY849), PLCγ (pY783), STAT3(pY705), Akt1/2/3 (pS473), ERK1/2 (pThr202/pThr204), and their non-phosphorylated counterparts, as well as α-tubulin as a loading control. The immunoblots were cropped for clarity. Full length blots are presented in Figure S1. (b-e) Quantification of phosphorylated proteins relative to total proteins.
Phosphorylation at 30 min of stimulation with PDGF-AA was set as 1. The experiment is a representative one out of three experiments performed withsimilar results. (f, g) Tet-inducible PAE cells were seeded into 96 well plates, incubated in Ham’s F-12 media supplemented with 1% FBS, induced or notwith doxycycline, and then stimulated with 0, 1, 5, 10 or 20 ng/ml of PDGF-AA (f) or PDGF-BB (g) for 72 h. Cells incubated in media with 10% FBS wereused as a positive control. WST-1 was added into the wells for 4 h and the absorbance of cells incubated in 1% FBS without doxycycline and PDGF ligands
was set as 1. The results from four independent repeats are plotted. The statistically significant difference between WT and K917R mutant PDGFRα tetinducible PAE cell lines was determined by unpaired, two-tail student t-test. *p < 0.05

(Fig. 7c). The background phosphorylation of PLCγ in
K917R PDGFRα cells, before stimulation with PDGF-AA,
was reproducibly decreased compared to WT PDGFRα,
whereas the background phosphorylation of STAT3 wasincreased. This was a consistent finding, the explanation
for which remains to be determined. However, activationof other signaling molecules, including ERK1/2 MAPkinase (Fig. 7d) and Akt, a downstream effector of PI3K
(Fig. 7e), were not affected. Similar results were obtainedwith tet-inducible stable cell lines (data not known).

Effects of mutation of K917 on cell migration and cellproliferation

It has been reported that SUMOylation may affect cell
migration [29–31] and cell proliferation [32, 33]. In viewof our finding of altered activation of signaling pathways
downstream of PDGFRα, we analyzed whether thesechanges had functional effects on the cells, but have notfound any effect of K917R mutation on PDGFRα-induced
migration (data not shown). The effects on cell proliferation in response to different concentrations of PDGF-AAand PDGF-BB, were determined using WT and K917R
mutant PDGFRα tet-inducible PAE cell lines. The cells

Wang et al. BMC Molecular and Cell Biology(2023) 24:19

treated with doxycycline responded to both PDGF-AA(Fig. 7f ) and PDGF-BB (Fig. 7g) stimulation. The proliferative response of cells expressing K917R mutant PDGFRαto ligand stimulation was inhibited compared with cells
expressing the WT receptor, especially when cells were
stimulated with PDGF-BB (Fig. 7g).

Discussion
SUMOylation of PDGFRα at lysine residue 917 wasrecently reported in a mass spectrometry analysis [20].
In the present study, we confirmed SUMOylation of
PDGFRα using immunoblotting, while no SUMOylationof PDGFRβ was detected. Upon PDGF-AA stimulation, SUMOylation of PDGFRα and associated proteinspeaked at 45 min when the receptor was overexpressedin COS-7 cells, while phosphorylation and ubiquitinationof PDGFRα were seen after 5 min. Introduction of theinhibitor of the SUMO E1 enzyme SAE1/SAE2 ginkgolicacid attenuated SUMOylation of PDGFRα. Interestingly,
under denaturing conditions, less SUMOylation wasobserved upon PDGF-AA stimulation in RPE1 cells, suggesting that, apart from PDGFRα, other proteins binding to PDGFRα may also be SUMOylated. SUMOylationof PDGFRα was detected in RPE1 cells that were notstimulated with PDGF, indicating that some backgroundlevel of SUMOylation exists in growing cells. It has beenfound that glucose- and serum-starvation promote REV1
SUMOylation [34]. On the other hand, reduced SF-1
SUMOylation under serum-starvation was reported[35]. Therefore, we investigated the SUMOylation of
PDGFRα under serum-starvation versus non-starvation
conditions. We observed more SUMOylated PDGFRαunder serum-starvation in COS-7 cells (data not shown),
while the reverse was true for RPE1 cells. These results
indicated that the effects of starvation on PDGFRαSUMOylation may vary in different cell lines.
The crosstalk between SUMOylation and ubiquitination is complex. Protein SUMOylation and ubiquitination
can occur on the same lysine residues of their substrates,
which might lead to competition between SUMOylation
and ubiquitination, in which case SUMOylation may stabilize its target protein by inhibiting its ubiquitination.
For example, SUMOylation of lysine residue 21 of IκBα
blocks the ubiquitination at the same site, thus stabilizing IκBα [36]. SUMOylation has also been reported tofacilitate subsequent ubiquitination of the substrates via
STUbLs [37]. In the present study, we noticed a lower
degree of ubiquitination of the K917R mutant PDGFRαcompared with WT PDGFRα, which may suggest that
this site can be both ubiquitinated and SUMOylated.
Alternatively, SUMOylation of lysine residue 917 maypromote ubiquitination of other lysine residues in thereceptor.

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SUMOylation has been reported to increase protein
stability [22, 38]. Since our results showed less ubiquitination of the K917R mutant PDGFRα, we investigatedthe degradation and internalization of receptors which
are usually promoted by ubiquitination [19]. However, wefound no difference in the steady levels or ligand-induced
degradation between WT and K917R mutant PDGFRα.
Surprisingly, we also did not notice any influence of theK917R mutation on the internalization of the receptor from the cell membrane upon PDGF-AA stimulation, as well as on the trafficking of the PDGFRα to earlyand late endosomes. SUMOylation has been found to
be related to the localization of receptors to the nucleus[8], cilia [39], and Golgi [10]. However, we could notdetect any ciliary or nuclear localization of PDGFRα (notshown) and did not notice any difference in the localization between WT and K917R mutant PDGFRα to otherorganelles.
Posttranslational modifications of RTKs, including
ubiquitination and SUMOylation, may affect their downstream signaling. Our study showed delayed activationof PLC-γ and enhanced activation of STAT3 in K917R
mutant PDGFRα cells after ligand stimulation. In ourprevious research, we found that USP17 and USP4 deubiquitinated PDGFRβ and increased the level of activated
STAT3 [40]. The present study showed reduced ubiquitination of K917R mutant PDGFRα, which correlated toincreased STAT3 activation. This is consistent with theprevious finding and suggests that the level of ubiquitination of PDGFRα also may regulate the downstream
signaling rather than simply targeting the receptor forproteasomal and/or lysosomal degradation. Since we
did not observe any effect of SUMOylation on stability, trafficking or degradation of PDGFRα, it is possiblethat SUMOylation of PDGFRα affects interactions withdownstream effector molecules or their phosphorylation,
leading to effects on cell proliferation.
It has been known that abolition of SUMOylation dramatically reduced cell proliferation mediated by Akt1
[33]. Interestingly, we noticed less proliferation of PAE
cells induced to express K917R mutant PDGFRα thancells induced to express WT PDGFRα, especially whencells were stimulated with PDGF-BB. The reason forthe differences in the proliferative response of wild-type
PDGFRα expressing cells to PDGF-AA and PDGF-BB
remains to be determined, but could be due to the factthat PDGF-AA is a less potent activator of PDGFRαthan PDGF-BB in the PAE cell line. A contribution fromendogenous PDGFRβ is unlikely, since PAE cells do not
express PDGFRβ and there was no effect of PDGF-BB
stimulation on non-induced PAE cells.
In conclusion, by mutating lysine residue 917 of
PDGFRα to an arginine residue, we investigated thefunctional role of SUMOylation of this site on receptor

Wang et al. BMC Molecular and Cell Biology(2023) 24:19

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function. We observed some effects on signaling and cellproliferation, however, overall the effects seen were mild.
It is possible that there are other SUMOylated residues,in addition to lysine 917, which may have contributed to
SUMO-dependent effects in the K917R mutant PDGFRα,possibly involving multiple SUMO enzymes [41].

Ultracompetent Cells provided in the kit following themanual. The plasmids were isolated and sequenced toconfirm the presence of mutation. Wild-type (WT) andK917R mutant PDGFRα constructs were further used fortransient transfection and to create stable cell lines.

Conclusions
In summary, we have identified PDGFRα as a
SUMOylation substrate, and have presented an initialcharacterization of the functional role of SUMOylationof PDGFRα. Further studies on the effect of PDGFRαSUMOylation on receptor regulation and cellular effects
remain to be performed.

African green monkey kidney fibroblast-like cells COS-7(purchased from ATCC, CRL-1651), human embryonickidney (HEK) 293 cells (purchased from ATCC, CRL1573), and human retinal pigment epithelial-1 (RPE-1)
cells (kindly provided by Soren Christensen, University of
Copenhagen, Denmark; ATCC, CRL-4000) were cultured
in Dulbecco’s modified Eagle medium (DMEM) (SigmaAldrich), supplemented with 10% fetal bovine serum(FBS) (Biowest). Porcine aortic endothelial (PAE) cells
were cultured in Ham’s F-12 Nutrient Mix, GlutaMAX™
Supplement (Thermo Fisher Scientific) containing 10%
FBS. WT and K917R mutant PDGFRα stable inducible
cell lines were cultured in DMEM media, supplemented
with 0.8 µg/ml puromycin and 10% FBS. For starvation,
cells were incubated in DMEM medium supplemented
with 0.1% FBS for 24 h. Cells were transiently transfectedwith plasmids using Lipofectamine 3000 reagent (Invitrogen), following the protocol provided by the vendor.

Materials and methods
Reagents

An antibody against PDGFRα (AF 1602) was purchasedfrom R&D Systems (Minneapolis, MN, USA). The antibodies recognizing phospho-PDGFRα (pY849, #3170),
phospho-PLCγ1 (pTyr783, #2821), PLCγ1 (#2822),
phospho-STAT3 (pTyr705, D3A7, #9145), STAT3(79D7, #4904), phospho-Akt (pSer473, D9E, #4060),
Akt (#9272S), phospho-ERK1/2 (pThr202/pThr204,#9101) and ERK1/2 (137F5, #4695) were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against SUMO1 (sc-5308) and ubiquitin (sc-8017)
were from Santa Cruz Biotechnology (Santa Cruz, CA,
USA). The α-tubulin antibody (T6074) was from SigmaAldrich (St Louis, MO, USA). Primary antibodies against
HA (NB600-363) and the transferrin receptor (TfR)(NB100-92243) were from Novus Biologicals (Centennial, CO, USA). Antisera recognizing Alix (HP95) [42]and PDGFRβ (CTβ) [43] were prepared in house. The
secondary antibodies used for immunoblotting included
goat anti-mouse IgG (62-6520), goat anti-rabbit IgG(65-6120) and rabbit anti-goat IgG (81-1620), were from
Invitrogen (Waltham, MA, USA). The inhibitors MG132(C2211) and chloroquine (C6628) were from SigmaAldrich (St Louis, MO, USA). The inhibitor ginkgolicacid (345,887) was purchased from Merck KGaA andbortezomib (#2244) was from Cell Signaling Technology(Danvers, MA, USA).
Mutagenesis

QuikChange Lightning Site-Directed Mutagenesis Kit(Agilent Technologies, 210,518) was used to mutate
lysine residue 917 of PDGFRα to an arginine residue.
Thermal cycling for PCR was performed using plasmid
pcDNA3-PDGFRα as the template and primers containing the mutation were designed by the online tool
(https://www.chem.agilent.com/store/primerDesignProgram.jsp). The amplification products were then digested
by Dpn I enzyme and transformed into XL10-Gold

Cell culture and plasmid transfection

Cell lysis, immunoprecipitation and immunoblotting

After various treatments, cells were washed once withphosphate-buffered saline (PBS), pre-chilled at 4℃
and then lysed in RIPA buffer (1% NP-40, 0.5% sodium
deoxycholate, 10% glycerol, 20 mM Tris-HCl, pH 7.5,
150 mM NaCl), supplemented with 1 mM Pefa Block,
1 mM sodium orthovanadate (Na3VO4) and 20 mM
N-ethylmaleimide (NEM), on ice for 5 min. The lysates
were centrifuged at 13,000 rpm for 15 min at 4℃ and thesupernatants were collected. To prepare cell lysates for
sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE), 20 µl of 6× SDS sample buffer (500 mM Tris-HCl, pH 6.8, 30% glycerol, 10% SDS,
600 mM dithiothreitol, 0.01% bromophenol blue) was
added to 100 µl of the supernatants. For immunoprecipitation, antibodies were added to the supernatants
at a final concentration of 0.2 µg/ml and samples wereincubated overnight at 4℃ with end-over-end rotation;
Dynabeads Protein A/Protein G (Invitrogen) was thenadded and incubation was prolonged for one more hour.
The beads were washed with lysis buffer three times andheated in SDS sample buffer at 95℃ for 3 min, followed
by SDS-PAGE and immunoblotting. After electrophoresis, proteins were electro-transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon P). The
membranes were blocked in PBS containing 0.1% Tween
20 (PBST) and 5% bovine serum albumin (BSA) for 1 h,
incubated with primary antibodies diluted in PBST

Wang et al. BMC Molecular and Cell Biology(2023) 24:19

containing 1% BSA overnight at 4℃, washed threetimes in PBST for 5 min each time, incubated in peroxidase-conjugated secondary antibodies for 1 h, and thenwashed three times in PBST. Proteins were visualized
by SuperSignal West Dura Extended Duration Substrate(Thermo Scientific) and a charge-coupled device (CCD)
camera (Bio-Rad). Image Lab (Bio-Rad) was used toquantify the bands.
Biotinylation of cell surface PDGFRα

After starvation and stimulation, cells were washedonce with pre-chilled PBS, incubated in 0.3 mg/ml EZLink Sulfo-NHS-SS-Biotin (Thermo Scientific) in PBS
for 1 h on ice, blocked with 50 mM Tris-HCl, pH 7.4 for
5 min, washed once with PBS, and lysed in RIPA buffer
for 5 min on ice. After centrifugation at 13,000 rpm for
15 min at 4℃, prewashed Pierce Streptavidin Agarose(Thermo Scientific) was added to the supernatant andincubated end-over-end for 1 h at 4℃, followed by threetimes washes in lysis buffer and heating at 95oC in SDS
sample buffer.
Establishment of tet-inducible cell lines

Lenti-X Tet-One Inducible Expression System (Takara
Bio USA) was used to establish tet-inducible cell lines.
In-Fusion HD Cloning Kit (Takara Bio USA) was used
to clone WT or K917R mutant PDGFRα into the pLVXTetOne vectors and lentiviral particles were produced
using Lenti-X Packaging Single Shots (VSV-G), containing transfection reagent and lentiviral packaging plasmids. 293T cells were transfected with indicated plasmids
and lentiviral particles were harvested 24- and 48-hours
post-transfection. The collected medium was diluted
twice with F-12 medium containing 10% FBS and added
to a 6-well plate with PAE cells supplemented with 8 µg/
ml polybrene. After 16 h, the medium was replaced withDMEM medium supplemented with 10% FBS and 0.8 µg/
ml puromycin. The surviving PAE cells were cultured in
medium with puromycin and induced with 100 ng/ml
doxycycline to express WT or K917R mutant PDGFRα.
Cell proliferation assay

Single clones selected from tet-inducible PAE cells wereseeded into 96 well plates at a density of 500 cells per welland cultured for 24 h. The media was replaced with 100 µl
of F-12 media supplemented with 1% FBS and 1–20 ng/
ml PDGF-AA or PDGF-BB in the absence or presence of
100 ng/ml doxycycline, while 10% FBS was used as a positive control. After incubation for 3 days, 10 µl of WST-1
reagent (Roche, 11,644,807,001) was added into the well,incubated at 37℃ in 5% CO2 for 4 h. The absorbance wasthen measured using an ELISA reader at 440 nm, while
650 nm was used as the reference wavelength.

Page 11 of 12

Statistical analysis

Data were analyzed with Microsoft Excel or GraphPad
Prism. The values are shown as mean ± standard deviation for experiments with more than three repeats. The
statistical significance was determined by unpaired, twotail student t-test. *p < 0.05.

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12860-023-00481-6.
Supplementary Material 1
Acknowledgements
We thank Soren Christensen from University of Copenhagen for kindly
providing RPE-1 cells.
Authors’ contributions
KW performed essentially all experiments and wrote the first version of themanuscript. NP supervised the work and critically read the manuscript. AH
contributed to performing the experiments. JL supervised the work andrevised the manuscript. CHH conceived the idea, financed and supervised thework, and revised the manuscript.
Funding
The work was supported by the Swedish Cancer Society (grant no 190066 Pj
01 H) and China Scholarship Council.
Open access funding provided by Uppsala University.
Data Availability
The reagents used and datasets analyzed in the current study are available
from the corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 29 December 2022 / Accepted: 5 May 2023

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