Protein phosphorylation: A potential target in glioma development

Abstract Glioma is one of the most common primary brain tumors, and mortality due to this disease is second only to cardiovascular and cerebrovascular diseases. In traditional surgery, it is difficult to eradicate glioma; often recurrence increases its malignant degree, leading to a large number of patients killed by this disease. It is one of the most important subjects to study its pathogenesis and explore effective treatment methods. Research on glioma mechanisms mainly focuses on the effect of DNA methylation in epigenetics. Although there are many studies on protein phosphorylation, there is no overall regulatory mechanism. Protein phosphorylation regulates a variety of cell functions, such as cell growth, division and differentiation, and apoptosis. As a consequence, protein phosphorylation plays a leading part in various activities of glioma, and can also be used as a target to regulate the development of glioma. This review is aimed at studying the effect of protein phosphorylation on glioma, understanding the pathological mechanism, and an in‐depth analysis of it. The following is a discussion on glioma growth, migration and invasion, resistance and death in phosphorylation, and the possibility of treating glioma by phosphorylation.

According to the histopathological and clinical characteristics, there are four grades of glioma (I, II, III, and IV).Grade I is benign glioma.The second grade is between the benign and malignant boundary of the glioma.Grade III above belongs to malignant glioma.Malignant gliomas grow fast and lead to more severe brain edema.There is no exactly effective treatment for glioma.In spite of temozolomide (TMZ) chemotherapy, which increases long-term survival, treatment failure, and rapid glioma recurrence remain common. 1 Moreover, despite undergoing the comprehensive treatment of surgery, radiotherapy, and chemotherapy, low-level glioma is isolated from the normal brain region from mechanism and vascular mechanism.It makes the peripheral normal brain area denervation regulation and no longer be adjusted by the normal regulation.What is worse is that high-grade glioma patients have a shorter survival period, with an average survival period of less than 1 year.Glioma is also divided into oligodendroglioma, astrocytoma, medulloblastoma, glioblastoma (GBM), ependymoma, and choroid plexus papilloma.GBM is the most common one, which is a Grade IV astrocytoma.Owing to its aggressiveness, it cannot be completely resected, 2 and has a high mortality rate. 3

| Protein phosphorylation
Phosphoric acidification of protein is very common.Protein phosphorylation is necessary for the initiation of many biological phenomena, including cell growth, proliferation, apoptosis, and so on, because phosphorylation can directly regulate various aspects of protein function. 4,5Regulated strictly by protein phosphatases and protein kinases, once the regulation of protein phosphorylation is disordered, it often leads to serious diseases, for example, cancer and glioma. 6ltering phosphorylation in any of the proteins is possibly connected with glioma, and they could be used as the potential targets for drug development for the treatment of glioma.Thus, drugs that target the phosphorylation pathway represent a hopeful area for glioma therapy.The literature on protein phosphorylation and glioma has been screened on PubMed (Figure 1).The occurrence of glioma is the cause.If we control the occurrence, the glioma will decrease.KDM1A stabilization is related to the occurrence of glioma.Glycogen synthase kinase-3β (GSK3β) phosphorylates KDM1A serine 683 (Ser683) upon priming by CK1α, leading to the stabilization of KDM1A, which, in turn, leads to cancer stem cell self-renewal and GBM tumorigenesis. 7Polarized M2 macrophages enhance the phosphorylation of PGK1 threonine 243 (Thr243) by secreting interleukin-6.
In addition, inhibition of PGK1 T243 phosphorylation abolishes macrophage-promoted tumorigenesis and glycolysis. 8Therefore, phosphorylation of PGK1 T243 may also be connected with the prognosis and malignancy of GBM.It has been confirmed that Atoh1 reduced the incidence of glioma and extended survival.Tyrosine 78 undergoes phosphorylation probably due to a Jak2mediated pathway.Phosphorylation of tyrosine 78 makes Atoh1 more stable and enhances the transcriptional activity of Atoh1. 9To sum up the above, restraining phosphorylation of Jak2-mediated tyrosine 78 could reduce significantly glioma occurrence.EGFR/SRC/ ERK signaling stabilizes YTHDF2 protein by phosphorylating YTHDF2 Ser39 and Thr381 to promote tumorigenesis of GBM cells. 10The phosphorylation of KDM1A Ser683, PGK1 T243, Atoh1 tyrosine 78, YTHDF2 Ser39, and Thr381 can promote tumorigenesis, and if we can effectively control the phosphorylation of these proteins, we can prevent the development of glioma.Related literature is shown in Table 1.

| The growth of the glioma
After reading a lot of related literatures, I have listed a few briefly, from which we can find the relevance of phosphorylation and glioma growth.Phosphorylation has an influence on the size of glioma.A small-molecule inhibitor (Lck-I) was used to inhibit Lck phosphorylation, which blocked the phosphorylation of Paxillin and Crk-II.Lck-I could be used to treat human glioma stem cells, and it significantly inhibited self-renewal, tumorsphere formation, and glioma size. 11Overexpression of SAE1 induces increased SUMOylation and Ser473 phosphorylation of AKT, which stimulates the growth of glioma cells in vitro and in a nude mouse tumor model. 12Src phosphorylation has a positive role in the growth rate of glioma cells.Moreover, several tumor cell growth inhibitors decrease autophosphorylation of FAK and delay the GBM cell cycle progression. 13The phosphorylation of PFK1 platelet isoform (PFKP) S386 not only increased its expression but also promoted aerobic glycolysis and brain tumor growth. 14limination of TRIP13 Y56 phosphorylation significantly reduced epidermal growth factor receptor (EGFR) signaling and GBM cell growth. 15The network of the above growthrelated regulatory proteins was analyzed by the STRING online platform (Figure 2A).The results show that, except TRIP13, all the above proteins were directly or indirectly related, especially ATK1, which was closely related to other proteins.Moreover, the above regulatory protein phosphorylation has positive significance for the size, morphology, and growth rate of glioma, so we can achieve the effect of limiting glioma growth by inhibiting the phosphorylation of one or more of the above proteins.Related literature is shown in Table 2.

| The proliferation of glioma
All cells can proliferate, hence we need to prevent the proliferation of glioma cells to slow the progression of the tumor.PFKP S386 phosphorylation could increase cell proliferation. 14Eukaryotic cells generally proliferate by mitosis.Regulation of Olig2 mitosis and glioma function requires phosphorylation of three serine motifs (S10, S13, and S14) at the amino terminus. 16PKMYT1 and WEE1 restrain cell cycle protein B-CDK1 activity through the phosphorylation of CDK1-Y15 and accelerate timely completion of mitosis in GBM stem-like cells (GSCs). 17cycle arrest and slow down the proliferation rate of glioma cells. 18Similarly, MiR-29a/b/c induces G1 blockade by blocking AKT phosphorylation to the extent that it inhibits tumor cell proliferation. 19Therefore, phosphorylation is closely associated with mitosis in glioma cells.Fasudil, a ROCK2 phosphorylation inhibitor, significantly inhibited the proliferation of TMZ-resistant gliomas. 20STAT3 tyrosine phosphorylation has been proved to accelerate the growth and glioma cell proliferation ensuing in significant long-term survival.2][23] Knockout of TRIM22 decrease IKKα/β and IκBα phosphorylation and reduce GBM cell proliferation. 24The phosphorylation of PFKP, serine motif (S10, S13, and S14), CDK1, Akt, ROCK2, STAT3, IKKα/β, and IκBα could improve glioma proliferation.According to the protein interaction network, we know that all the proteins except ROCK2 have a strong interactive relationship (Figure 2B).To sum up, we can inhibit glioma mitosis and proliferation rate by adapting protein phosphorylation.Related literature is shown in Table 3.

| Phosphorylation and glioma migration and invasion
Inside the brain, glioma cells migrate rapidly according to the extracellular environment and invade surrounding structures.The reason why N-acetylgalactosaminyltransferase 2 (GALNT2) was closely associated with the migration and invasion of glioma cells is that GALNT2 facilitated the malignant features of gliomas by affecting the O-glycosylation, EGFR phosphorylation, and subsequently the downstream PI3K/Akt/mTOR axis. 25Thus, GALNT2 knockout decreases the level of phosphorylated EGFR, which could improve the malignant characteristics of glioma.Hence GALNT2 may serve as a potential target of action to treat glioma in the future.Small interfering RNA-mediated WNK1 or OSR1 knockdown reduced glioma migration by eliminating NKCC1-regulated phosphorylation activation. 26Similarly, RELN regulated the migration of GBM cells through DAB1 tyrosine phosphorylation. 27The major feature of GBM is the invasion of the healthy brain parenchyma.The invasion of glioma is always what we value most.Heparin-binding EGF (HB-EGF) promoted EGFRvA Y845 site and STAT3 phosphorylation, which generated a positive feedback loop and it may enhance invasive function. 28Actomyosin is closely related to the invasive function of glioma.Myosin light chain 2 expression and phosphorylation Yang et al. 12

SAE1 expression level, SUMOylation, and Ser473
Regulate SAE1 Both SAE1 overexpression and silencing induced a corresponding increase or decrease in the phosphorylation of SUMOylation and Ser473, thereby promoting or inhibiting glioma cell growth.

Cell viability
Several tumor cell growth inhibitors Reduces autophosphorylation of FAK, halts cells in the G2/M phase of the cell cycle, and delays the progression of cell growth and proliferation.
Hu et al. 15 6.5080 GBM cell growth Abrogating TRIP13 Y56 phosphorylation Dramatically attenuate EGFR signaling and GBM cell growth.
Lee et al. 14
T A B L E 3 Literature shows that protein phosphorylation affects the proliferation of glioma

Reference
Impact factor

Fluency indices
Intervening measure
Zhou et al. 16 7.8150 Phosphorylation of S10, S13, and S14 Nothing Phosphorylation of amino-terminal S10, S13, and S14 regulates the mitosis of Olig2 and also controls the growth of glioma cells.
Toledo et al.Teng et al. 33 10.0910 EphB1 and EphB1 phosphorylation Overexpress EphB1 EphB1 overexpression did not affect external cell migration and invasion.However, EphB1 phosphorylation decreased migration and invasion.
are closely connected to MerTK activity, suggesting that the role of MerTK in glioma cell invasion is mediated through the contractility of actin.The receptor MerTK overexpressed and increased invasive potential in GBM. 29KAP low expression activates Cdk2, thereby promoting invasion through increased retinoblastoma phosphorylation and inactivation of the actin inhibitors mediated by Cdc2. 30Chronophin protein expression has an inverse association with phosphorylation level of cofilin in glioma cells.Chronophin-deficient GBM cells revealed elevated cofilin phosphorylation, increased polymeric actin, and higher cell migration directionality, and they improved in vitro invasiveness. 31Besides, phosphorylation of proline-rich tyrosine kinase 2 (Pyk2) is involved in the mechanism of store-operated Ca (2+) entry (SOCE) regulating adhesive foci transformation and epithelial-mesenchymal (like) transformation in glioma cells. 32What can be seen from this is that the SOCE-Pyk2 pathway is essential for migration and invasion of glioma.Ephrin-B2/Fc-induced phosphorylation of EphB1 significantly reduced migration and invasion. 33In addition, a class of glioma cells has unphosphorylated OLIG2 (S10, S13, and S14), yet these cells are highly migratory and invasive in vitro and in vivo.The mechanism is that unphosphorylated OLIG2 induced TGF-β2 expression and promoted the aggressive mesenchymal properties of glioma cells. 34rotein-protein interaction network of the aboverelated regulatory proteins was built (Figure 2C).When phosphorylated, each protein can promote or restrain glioma migration and invasion.Therefore, we can regulate glioma migration and invasion through protein interactions.Related literature is shown in Table 4.

| Phosphorylation and the resistance of glioma
Glioma resistance could make chemotherapy fail.Current treatments also hope to bypass the resistance of tumor cells, thereby achieving a breakthrough.ACT001, originally an ancient anti-inflammatory drug, was found to demonstrate anti-GBM activity in clinical trials.ACT001 treatment can modulate antitumor immunity in GBM by inhibiting phosphorylated STAT3. 35Thus, STAT3 has a significant impact on glioma immunosuppression.The mechanism involved is that the tyrosine kinase receptor Tie2 physically interacted with FGFR1 to increase drug resistance expression in GSCs by promoting STAT3 phosphorylation and binding to the AURKA promoter. 36We can influence the immunity of   | 183 glioma via phosphorylation.A combined ERK/mTOR targeted elimination inhibits GSK-3β phosphorylation, leading to MAP1B phosphorylation and confers cellular sensitization. 37The above data represented molecular signaling networks for chronic inhibition of anti-mTOR in cultured primary human GBM cells and point to novel therapeutic strategies.Plus, we can also reduce sensitivity.A novel N-linked glycosylation inhibitor (NGI-1) inhibited multiple receptor tyrosine kinase glycosylation and phosphorylation in GBM.NGI-1 sensitized GBM in multiple models to chemotherapy and radiotherapy for therapeutic purposes and may be particularly effective in GBMs with preserved PTEN activity. 38Targeted therapy against PI3K has become a very popular treatment for GBM; nevertheless, the emergence of resistance limited treatment potential.Thus, to achieve a good therapeutic effect, the resistance of glioma can be affected by regulating protein phosphorylation.Related literature is shown in Table 5.

| Phosphorylation and the death of glioma cell
The death of glioma cells includes apoptosis and necrosis.Glioma cell death often interferes with the experiment.PLK4 interacted with IKBKE and thus phosphorylated it, resulting in an increase in NF-κB antiapoptosis in GBM cells. 39SAE1 silence obviously suppressed the phosphorylation of Akt SUMOylation and Ser473, induced a G2 phase block in the cell cycle, and led to apoptosis driven by a series of biochemical molecular events. 12ANXA2 increased the expression of miR155HG through STAT3 phosphorylation.Similarly, knocking down miR155HG induced the G1/S phase block, which, in turn, inhibited division and increased apoptosis. 40GBM 8401 cells transfected with wild-type (WT) IKKα restrained zerumbone-induced apoptosis as well as significantly reduced the levels of IKKα phosphorylation in a time-dependent manner.
Similarly, transfection of GBM 8401 cells with Akt showed a similar effect. 41Therefore, the conclusion is that followed by Akt and IKKα phosphorylation was involved in zerumbone-induced apoptosis in GBM cells.Downregulation of cathepsin L resulted in a significant decrease in CD133 expression and a decrease in phosphorylation of ATM and DNA-PKcs.Cathepsin L inhibition significantly reduced GSC growth and promoted apoptosis. 42Thus, it can be seen the phosphorylation of ATM and DNA-PKcs is associated with cell apoptosis.Autophagy is a pretty typical form of death.Inhibition of AKT-MTOR-RPS6KB1 pathway induced GBM cells autophagy according to phosphorylation status. 43MST4 phosphorylated serine 383 of ATG4B, activated ATG4B, and increased cellular autophagic flux. 44Besides, if MDA-9 is inhibited, GSCs will undergo cellular autophagic and die.MDA-9 suppresses high levels of autophagy by phosphorylating BCL2 and EGFR signaling to achieve self-protection. 45,46Besides, regarding autophagy, PAK1-mediated phosphorylation of ATG5 and phosphorylation of c-MET and PI3K in GPCs under hypoxia conditions could affect cell autophagy. 47,48There was an experiment that revealed that the phosphorylation level of p62/SQSTM1 was reduced in the presence of PIM knockdown.However, phosphorylated p62/ SQSTM1 (S332E) abolished the effect of PIM inhibitors and the upregulation of apoptosis on the apoptosisinducing ligand (TRAIL)-r2/DR5. 49Globally, it represents that p62/SQSTM1 (Ser332) phosphorylation regulates the TRAIL-activated external apoptotic pathway and thus controls apoptosis.PKM2 also regulates apoptosis.PKM2 in mitochondria phosphorylates the Bcl2 T69 site, and the level of phosphorylation and conformational changes correlate with the grading and prognosis of malignant gliomas. 50It revealed a mechanism of effect that can directly inhibit apoptosis through phosphorylation of Bcl2.Moreover, circ-MAPK4 regulates apoptosis in glioma cells by regulating p38/MAPK phosphorylation level. 51Above experiments confirmed that phosphorylation can adjust the death of glioma cells to improve the success rate of the experiment.The network of the above death-related regulatory proteins was analyzed by STRING online platform (Figure 2D).The results showed that all the above proteins were directly or indirectly related.Thus, we can achieve the effect of controlling glioma death by regulating the phosphorylation of one or more of the above proteins.Related literature is shown in Table 6.

| Phosphorylation and other properties of glioma
Now let us observe other effects of protein phosphorylation on glioma.Tumor heterogeneity is an important feature of malignancy, which could significantly affect the therapeutic effects and prognosis.Glioma cells exhibited heterogeneous patterns of TGF-β pathway activation with altered phosphorylation of SMAD2, SMAD3, and SMAD1/5/8. 52Proteomics, closely related to protein phosphorylation, has been widely used in the field of heterogeneity research.In addition, the stemness of glioma is the basis of its survival and proliferation and is the key to play the Yang et al. 12 5.1110 SUMOylated proteins, Ser473 phosphorylation SAE1 overexpression or silence SAE1 expression positively correlates with phosphorylation of Akt SUMOylation and Ser473 and controls the growth and malignancy of gliomas.

miR155HG expression
Intracranial GBM mouse model; knock out miR155HG ANXA2 promoted phosphorylation of STAT3 to upregulate miR155HG expression.Knocking down miR155HG led to blocking of the G1/S phase, which elevated the chance of apoptosis.

Akt phosphorylation
Transfected IKK and Akt Zerumbone significantly prevented phosphorylation of Akt.Akt and FOXO1 phosphorylation facilitated zerumbone-mediated apoptosis in glioma cells.
Wang et al. 42 6.5080CD133 expression, phosphorylation of ATM and DNA-PKcs Set up control groups; knockdown of cathepsin L Reduce CD133 expression and decrease phosphorylation of ATM and DNA-PKcs.Histone L, combined with radiotherapy, can be targeted for inhibition and promotion of apoptosis.

Phosphorylation of p62/SQSTM1
Knock out PIM A decreased phosphorylation of p62/SQSTM1 and increased apoptosis.

Phosphorylation of Bcl2
Nothing Bcl2 phosphorylation inhibits apoptosis directly.
He et al. 51 10.6790 Phosphorylation levels of p38/MAPK Inhibiting miR-125a-3p The upregulation of p38/MAPK phosphorylation could be slowed down to the extent that circ-MAPK4-induced apoptosis in glioma cells did not increase excessively.
| 185 role of malignant biology.Studies have shown that hypoxia can enhance the stemness of gliomas.Hypoxia downregulated ID2 phosphorylation on Thr27 through inactivation of DYRK1A and DYRK1B.
In contrast, high DYRK1 expression increases ID2 Thr27 phosphorylation, leading to glioma stemness deficiency, which inhibits tumor growth and is more favorable to the prognosis of GBM patients. 53To sum up, protein phosphorylation has an influence on the heterogeneity and stemness of glioma.Other properties remain to be further studied.Related literature is shown in Table 7. Currently, many drug studies refer to protein phosphorylation.Similarly, various treatments for glioma are associated with protein phosphorylation.We just need to figure out that phosphorylation has an influence on glioma, and find a way to break through from the aspect.We all know that GALNT2 is inextricably linked to the physiological processes of cell proliferation, migration, and invasion in gliomas.But the mechanism by which GALNT2 promotes the malignant features of gliomas is by affecting EGFR phosphorylation. 25Concurrent inhibition of p110β and JNK restrains the phosphorylation of Akt, FAK, and zyxin to inhibit the proliferation and migration of GBM cells. 54Besides, Histone phosphorylation plays an important regulatory role in DNA repair structure and transcription during cell proliferation and apoptosis. 55One of the major tasks of phosphoproteins is to provide potential biomarkers for either diagnosis or drug targets in medical applications. 56STAT3 phosphorylation can regulate the proliferation, migration, invasion, apoptosis, and other physiological processes of glioma, thus STAT is a good regulatory target, and the targeted inhibition of glioma by STAT phosphorylation has a good therapeutic prospect.The proteins AKT and FGFR, which appeared frequently in this paper, have become important therapeutic targets for glioma and improved the sensitivity of glioma by targeted inhibition of phosphorylation, thus playing an active role in the treatment of glioma. 57Phosphorylation of many proteins can affect the development of glioma (Figure 3).To conclude, protein phosphorylation will be a breakthrough, providing new ideas for the treatment of glioma.Therefore, it is entirely possible to treat glioma by phosphorylation.

| CONCLUSIONS AND PROSPECTS
Protein phosphorylation is associated with the occurrence, growth, proliferation, migration, invasion, resistance, and death of glioma cells, while other properties of glioma cells remain to be further investigated.As is well known, phosphorylation and dephosphorylation of protein, which is a reversible process, almost regulate the cytoskeleton development and cell proliferation, differentiation, apoptosis, and all the process of life activity.Reversible protein phosphorylation is one of the main currently known ways of signal transduction.Therefore, we can effectively control glioma cells depending on protein phosphorylation.
We just need to grasp the corresponding mechanism of influence to find a method to the problem.If we can make good use of protein phosphorylation, glioma will also have pretty treatment, not limited to surgery.Although at present, the targeted therapy of intracellular signaling pathways has not gained a great satisfactory effect, we believe that the protein phosphorylation targeted regulation can achieve great effect in the future; its targeted drugs can also be further developed and put into use in the clinic, which provides another way for the treatment of patients and has a bright prospect.

F I G U R E 1 2 . 1 |
The literature screening flow chart of mechanisms by which phosphorylation affects glioma.N in the figure represents the number of literature.[Color figure can be viewed at wileyonlinelibrary.com]PHOSPHORYLATION AS A TARGET IN GLIOMA DEVELOPMENT Phosphorylation and the growth and development of glioma 2.1.1 | The occurrence of glioma Reduced cyclin D1 levels, selective downregulation of Akt Ser473 phosphorylation, and VEGF receptor levels in parkin-expressing glioma cells promote G (1) phase cell F I G U R E 2 The protein interaction network.(A) The protein interaction network of related regulatory proteins in which phosphorylation affects glioma growth.(B) The protein interaction network of related regulatory proteins in which phosphorylation affects glioma proliferation.(C) The protein interaction network of related regulatory proteins in which phosphorylation affects glioma migration and invasion.(D) The protein interaction network of related regulatory proteins in which phosphorylation affects glioma apoptosis and autophagy.[Color figure can be viewed at wileyonlinelibrary.com] Literatures shown the connection between protein phosphorylation and the growth of gliomaReference blocks the phosphorylation of Lck, which, in turn, inhibits the formation of pseudopods in glioma cells, thereby controlling not only migration but also the formation of tumorspheres.

T A B L E 5
Protein phosphorylation has an influence on the resistance of glioma Reference analysis, RT-PCR, and immunofluorescence Inhibiting phosphorylated STAT3 can modulate antitumor immunity in GBM.Li et al.

T A B L E 6
Protein phosphorylation could regulate the death of glioma of PLK4 inhibits cell growth, and PLK4 phosphorylation with IKBKE promotes NF-κB transcription and inhibits apoptosis.
phosphorylation of BCL2, and pBCL2 is downregulated, and it induced GSCs' death.Feng et al. 47 (2021) 10.091 Levels of PAK1 (K420) acetylation, ATG5 (T101) phosphorylation Hypoxia The two were significantly correlated.Hypoxia induced ELP3-mediated PAK1 acetylation and PAK1 induced phosphorylation of ATG5 to promote autophagy.Cheng et al.48 of c-MET and PI3K Hypoxic drug c-MET and PI3K phosphorylation in GPCs under hypoxic conditions was reduced by c-MET inhibitor, leading to oxidative mutagenesis and apoptosis.

T A B L E 7
Literatures shown the effect of protein phosphorylation on other aspects of glioma phosphorylation, decreases HIF2α stability, and inhibits glioma cell growth and loss of tumor stem cells.F I G U R E 3 The structural diagram of the mechanism by which phosphorylation affects glioma (+) represents the promoting effect of protein phosphorylation on the properties of glioma.(−) represents the inhibitory effect of protein phosphorylation on the properties of glioma.[Color figure can be viewed at wileyonlinelibrary.com]