Stomatin-like protein 2 (STOML2), 也被稱為SLP-2或paratarg-7，是一種粒線體內膜蛋白且對於粒線體功能十分重要，並在許多類型的癌症中扮演致癌基因的角色。STOML2在絲胺酸Ser17位點會受到PKC-ζ催化而造成的過度磷酸化，是漿細胞病變中體染色體顯性遺傳性的一個顯著風險因子，例如多發性骨髓瘤(multiple myeloma, MM)。本研究中我們探討STOML2的磷酸化對於STOML2的細胞內座落位置以及STOML2促進粒線體增生活性的影響。藉由phos-tag SDS-PAGE分析比較野生型STOML2 WT以及兩種丙胺酸取代突變型STOML2 S17A和STOML2 S21A，結果顯示在接近70 kDa的一個磷酸化STOML2異構型能夠代表STOML2絲胺酸Ser17的磷酸化。除了與先前文獻相符合的是共同表現PKC-ζ能夠增加STOML2 絲胺酸S17磷酸化的結果，而我們發現共同表現PKC-ζ並使用PKC活化劑PMA進行刺激亦能夠增加STOML2在Ser17的磷酸化。使用針對Flag的抗體進行對野生型Flag-STOML2 WT免疫螢光染色偵測的結果顯示，野生型Flag-STOML2 WT主要是以在細胞核周圍形成以大顆粒蛋白聚集體的型態，其次的型態包括大顆粒混合小顆粒蛋白聚集體的混合型態、點狀分散分布型態以及在細胞核周圍聚集型態。相較於野生型STOML2 WT，突變型Flag-STOML2 S17A和Flag-STOML2 S21A則減少了大顆粒蛋白聚集體的型態，並增加了混合型態以及點狀分散分布型態。共同表現PKC-ζ減少了野生型Flag-STOML2 WT呈現的大顆粒蛋白聚集體型態，增加了小顆粒及點狀分散分布型態，而過度表現PKC-ζ並使用PMA刺激下的變化類似於僅共同表現PKC-ζ。相反的，突變型Flag-STOML2 S17A和S21A的呈現型態並沒有受到共同表現PKC ζ及PMA刺激的影響。STOML2受到PKC刺激所引發的分子型態改變，和液體-液體相位分離(LLPS)相似。我們提出液體-液體相位分離是導致STOML2型態改變的機制的假設。利用軟體包括PONDR及dSCOPE對STOML2序列分析預測是否具液體-液體相位分離特性，結果顯示STOML2序列中具有可能液體-液體相位分離的特質，包括具有幾個無穩定構型及低複雜蛋白質序列區段，重要的是，這些區域中包括第1-31個胺基酸序列區段，並具有高度可能的液體-液體相位分離的特性，此區段包含了我們已進行點突變研究的絲胺酸S17及絲胺酸S21位點。再者，與此序列預測相符的是，我們使用能夠阻礙液體-液體相位分離的藥物1,6-hexanediol來處理細胞時，發現能夠顯著阻礙STOML2蛋白聚集體的形成。接著我們對藉由anti-Flag抗體的免疫螢光染色偵測到的野生型Flag-STOML2 WT、突變型STOML2 S17A及Flag-STOML2 S21A蛋白聚集體與粒線體進行共座落分析，發現這些STOML2聚集體與粒線體間的共座落欠佳(皮爾森相關係數0.4~0.5)，顯示STOML2蛋白聚集體主要分布於細胞質而非粒線體。但是當分析藉由anti-STOML2抗體的免疫螢光染色偵測到的野生型Flag-STOML2 WT、突變型Flag-STOML2 S17A及Flag-STOML2 S21A並進行共座落分析時，結果顯示皆與粒線體都有良好的共座落(皮爾森相關係數數值0.6~0.8)。相較於野生型Flag-STOML2 WT，突變型Flag-STOML2 S17A及S21A粒線體的共座落則有降低，其中STOML2 S17A具顯著統計意義的降低。此外，相較於野生型Flag-STOML2 WT，突變型Flag-STOML2 S17A及S21A對於增加粒線體質量增加的作用並無顯著差異。再者，我們發現增加PKC活性可增加野生型Flag-STOML2 WT對於促進粒線體質量增加的趨勢，但對於突變型Flag-STOML2 S17A及Flag-STOML2 S21A則無影響。綜合以上，我們的結果顯示，STOML2蛋白聚集體可能是經由含有 Ser17及Ser21的區段進行液體-液體液相分離所導致，且STOML2很可能在絲胺酸Ser17受PKC磷酸化時，調節液體-液體相位分離並促進STOML2增加粒線體增生的能力。

Stomatin-like protein 2 (STOML2), also known as SLP-2 or paratarg-7, is a mitochondrial inner membrane protein crucial for mitochondrial functions and acts as an oncogene in various types of cancer. Hyperphosphorylation of STOML2 at Ser17 catalyzed by PKC-ζ was the first autosomal-dominantly inherited risk factor for plasma cell diseases, such as multiple myeloma (MM). Here, we investigated the role of phosphorylation of STOML2 in regulating subcellular localization and mitochondrial biogenesis-promoting activity of STOML2. By comparing alanine substitution mutants, S17A and S21A, to wildtype (WT) STOML2 and phos-tag SDS-PAGE analysis, we identified a phospho-STOML2 isoform near 70 kDa corresponding to phosphorylated STOML2 at Ser17. In agreement with previous reports in the literature, co-expression of PKC-ζ increased phosphorylated STOML2 at Ser17, and co-expression of PKC-ζ and stimulation by PKC activator PMA further increased Ser17 phosphorylation. Immunofluorescence microscopy by anti-Flag antibody staining showed that exogenous Flag-STOML2 WT, hereafter referred to as STOML2 WT, protein predominantly displayed discrete large perinuclear aggregates, and to a lesser extent, a mix of both large perinuclear aggregates and perinuclear small puncta, disperse small puncta and perinuclear enrichment. Both Flag-STOML2 S17A and Flag-STOML2 S21A, hereafter referred to as STOML2 S17A and STOML2 S21A, displayed reduced amounts of discrete large perinuclear aggregates, and displayed increases in the mix type and disperse small puncta as compared with that of STOML2 WT. In addition, co-expression of PKC-ζ reduced amounts of large perinuclear aggregates of STOML2 WT but increased amounts of disperse small puncta. Co-expression of PKC-ζ along with PMA stimulation showed changes in the morphology of STOML2, similar to that of co-expression of PKC-ζ alone. In contrast, the expression pattern of both STOML2 S17A and STOML2 S21A was not affected by the co-expression of PKC-ζ or the co-expression of PKC-ζ and PMA stimulation. The change of molecular morphology of STOML2 by PKC activating is similar to liquid-liquid phase separation (LLPS). We hypothesized that LLPS is the mechanism underlying the shift of morphology of STOML2. Sequence analysis of STOML2 using web-based software for prediction of LLPS showed that STOML2 contains several intrinsically disordered and low complexity regions, and importantly, a region encompassing amino acid residue 1 to 31 with a high score for LLPS harbors S17 and S21, whose mutation affected PKC-mediated morphology shift of STOML2. In addition, treatment with 1-6 hexanediol, which blocks molecular hydrophobic interaction in LLPS, disrupted the formation of STOML2 WT aggregates. Colocalization analysis of Flag-STOML2 WT, Flag-STOML2 S17A, and Flag-STOML2 S21A stained by anti-Flag antibody showed that aggregates of Flag-STOML2 WT, Flag-STOML2 S17A, and Flag-STOML2 S21A were only modestly co-localized with mitochondria (Pearson’s correlation coefficient value 0.4~0.5), suggesting that aggregates of STOML2 are mainly cytosolic but not mitochondrial form. In contrast to the finding of cytosolic STOML2 aggregates stained by anti-Flag antibody, mitochondrial colocalization analysis of Flag-STOML2 WT, Flag-STOML2 S17A, and Flag-STOML2 S21A stained with anti-STOML2 antibody showed that STOML2 WT, STOML2 S17A, and STOML2 S21A were well colocalized with mitochondria with Pearson’s correlation coefficient value 0.6~0.8. However, both STOML2 S17A and S21A showed modestly reduced mitochondrial colocalization, and STOML2 S17A showed modestly but statistically significant decreased mitochondrial colocalization compared to that of STOML2 WT. In addition, no significant difference was found in the capability in increasing mitochondrial mass among Flag-STOML2 WT, Flag-STOML2 S17A, and Flag-STOML2 S21A. Moreover, increased PKC activity showed a trend to up-regulate the effect of STOML2 WT in increasing mitochondrial mass, but not STOML2 S17A and STOML2 S21A. Together, our data suggest that the formation of STOML2 aggregates may result from liquid-liquid phase separation through a region harboring Ser17 and Ser21 of STOML2, and PKC phosphorylation of STOML2, most likely through Ser17, may regulate phase separation and mitochondrial biogenesis-promoting activity of STOML2.

1. Lapatsina, L., Brand, J., Poole, K., Daumke, O., & Lewin, G. R. (2012). Stomatin-domain proteins. Eur J Cell Biol, 91(4), 240-245.
2. Christie, D. A., Lemke, C. D., Elias, I. M., Chau, L. A., Kirchhof, M. G., Li, B., Ball, E. H., Dunn, S. D., Hatch, G. M., & Madrenas, J. (2011). Stomatin-like protein 2 binds cardiolipin and regulates mitochondrial biogenesis and function. Mol Cell Biol, 31(18), 3845-3856.
3. Hajek, P., Chomyn, A., & Attardi, G. (2007). Identification of a novel mitochondrial complex containing mitofusin 2 and stomatin-like protein 2. J Biol Chem, 282(8), 5670-5681.
4. Da Cruz, S., Parone, P. A., Gonzalo, P., Bienvenut, W. V., Tondera, D., Jourdain, A., Quadroni, M., & Martinou, J. C. (2008). SLP-2 interacts with prohibitins in the mitochondrial inner membrane and contributes to their stability. Biochim Biophys Acta, 1783(5), 904-911.
5. Da Cruz, S., De Marchi, U., Frieden, M., Parone, P. A., Martinou, J. C., & Demaurex, N. (2010). SLP-2 negatively modulates mitochondrial sodium-calcium exchange. Cell Calcium, 47(1), 11-18.
6. Wang, Y., & Morrow, J. S. (2000). Identification and characterization of human SLP-2, a novel homologue of stomatin (band 7.2b) present in erythrocytes and other tissues. J Biol Chem, 275(11), 8062-8071.
7. Owczarek, C., Treutlein, H., Portbury, K., Gulluyan, L., Kola, I., & Hertzog, P. (2001). A novel member of the STOMATIN/EPB72/mec-2 family, stomatin-like 2 (STOML2), is ubiquitously expressed and localizes to HSA chromosome 9p13.1. Cytogenet Cell Genet, 92, 196 - 203.
8. Kirchhof, M. G., Chau, L. A., Lemke, C. D., Vardhana, S., Darlington, P. J., Márquez, M. E., Taylor, R., Rizkalla, K., Blanca, I., Dustin, M. L., & Madrenas, J. (2008). Modulation of T Cell Activation by Stomatin-Like Protein 2. J Immunol, 181(3), 1927-1936.
9. Tondera, D., Grandemange, S., Jourdain, A., Karbowski, M., Mattenberger, Y., Herzig, S., Da Cruz, S., Clerc, P., Raschke, I., Merkwirth, C., Ehses, S., Krause, F., Chan, D. C., Alexander, C., Bauer, C., Youle, R., Langer, T., & Martinou, J. C. (2009). SLP-2 is required for stress-induced mitochondrial hyperfusion. EMBO J, 28(11), 1589-1600.
10. Cui, Z., Zhang, L., Hua, Z., Cao, W., Feng, W., & Liu, Z. (2007). Stomatin-like protein 2 is overexpressed and related to cell growth in human endometrial adenocarcinoma. Oncol Rep, 17(4), 829-833.
11. Zhang, L., Ding, F., Cao, W., Liu, Z., Liu, W., Yu, Z., Wu, Y., Li, W., Li, Y., & Liu, Z. (2006). Stomatin-like protein 2 is overexpressed in cancer and involved in regulating cell growth and cell adhesion in human esophageal squamous cell carcinoma. Clin Cancer Res, 12(5), 1639-1646.
12. Wang, Y., Cao, W., Yu, Z., & Liu, Z. (2009). Downregulation of a mitochondria associated protein SLP-2 inhibits tumor cell motility, proliferation and enhances cell sensitivity to chemotherapeutic reagents. Cancer Biol Ther, 8(17), 1651-1658.
13. Christie, D. A., Kirchhof, M. G., Vardhana, S., Dustin, M. L., & Madrenas, J. (2012). Mitochondrial and plasma membrane pools of stomatin-like protein 2 coalesce at the immunological synapse during T cell activation. PLoS One, 7(5), e37144.
14. Preuss, K. D., Pfreundschuh, M., Ahlgrimm, M., Fadle, N., Regitz, E., Murawski, N., & Grass, S. (2009). A frequent target of paraproteins in the sera of patients with multiple myeloma and MGUS. Int J Cancer, 125(3), 656-661.
15. Preuss, K. D., Fadle, N., Regitz, E., Held, G., & Pfreundschuh, M. (2014). Inactivation of protein-phosphatase 2A causing hyperphosphorylation of autoantigenic paraprotein targets in MGUS/MM is due to an exchange of its regulatory subunits. Int J Cancer, 135(9), 2046-2053.
16. Preuss, K. D., Pfreundschuh, M., Fadle, N., Regitz, E., Raudies, S., Murwaski, N., Ahlgrimm, M., Bittenbring, J., Klotz, M., Schafer, K. H., Held, G., Neumann, F., & Grass, S. (2011). Hyperphosphorylation of autoantigenic targets of paraproteins is due to inactivation of PP2A. Blood, 118(12), 3340-3346.
17. Alberti, S., Gladfelter, A., & Mittag, T. (2019). Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates. Cell, 176(3), 419-434.
18. Wai, T., Saita, S., Nolte, H., Muller, S., Konig, T., Richter-Dennerlein, R., Sprenger, H. G., Madrenas, J., Muhlmeister, M., Brandt, U., Kruger, M., & Langer, T. (2016). The membrane scaffold SLP2 anchors a proteolytic hub in mitochondria containing PARL and the i-AAA protease YME1L. EMBO Rep, 17(12), 1844-1856.
19. Cao, W., Zhang, B., Liu, Y., Li, H., Zhang, S., Fu, L., Niu, Y., Ning, L., Cao, X., Liu, Z., & Sun, B. (2007). High-level SLP-2 expression and HER-2/neu protein expression are associated with decreased breast cancer patient survival. Am J Clin Pathol, 128(3), 430-436.
20. Zwick, C., Held, G., Auth, M., Bernal-Mizrachi, L., Roback, J. D., Sunay, S., Iida, S., Kuroda, Y., Sakai, A., Ziepert, M., Ueda, R., Pfreundschuh, M., & Preuss, K. D. (2014). Over one-third of African-American MGUS and multiple myeloma patients are carriers of hyperphosphorylated paratarg-7, an autosomal dominantly inherited risk factor for MGUS/MM. Int J Cancer, 135(4), 934-938.
21. Djunic, I., Elezovic, I., Ilic, V., Milosevic-Jovcic, N., Bila, J., Suvajdzic-Vukovic, N., Antic, D., Vidovic, A., & Tomin, D. (2014). The effect of paraprotein on platelet aggregation. J Clin Lab Anal, 28(2), 141-146.
22. Grass, S., Preuss, K.-D., Ahlgrimm, M., Fadle, N., Regitz, E., Pfoehler, C., Murawski, N., & Pfreundschuh, M. (2009). Association of a dominantly inherited hyperphosphorylated paraprotein target with sporadic and familial multiple myeloma and monoclonal gammopathy of undetermined significance: a case–control study. The Lancet Oncol, 10(10), 950-956.
23. Grass, S., Preuss, K. D., Wikowicz, A., Terpos, E., Ziepert, M., Nikolaus, D., Yang, Y., Fadle, N., Regitz, E., Dimopoulos, M. A., Treon, S. P., Hunter, Z. R., & Pfreundschuh, M. (2011). Hyperphosphorylated paratarg-7: a new molecularly defined risk factor for monoclonal gammopathy of undetermined significance of the IgM type and Waldenstrom macroglobulinemia. Blood, 117(10), 2918-2923.
24. Yang, C. T., Li, J. M., Li, L. F., Ko, Y. S., & Chen, J. T. (2018). Stomatin-like protein 2 regulates survivin expression in non-small cell lung cancer cells through beta-catenin signaling pathway. Cell Death Dis, 9(4), 425.
25. Liu, Q., Li, A., Wang, L., He, W., Zhao, L., Wu, C., Lu, S., Ye, X., Zhao, H., Shen, X., Xiao, X., & Liu, Z. (2020). Stomatin-like Protein 2 Promotes Tumor Cell Survival by Activating the JAK2-STAT3-PIM1 Pathway, Suggesting a Novel Therapy in CRC. Mol Ther Oncolytics, 17, 169-179.
26. Zheng, Y., Huang, C., Lu, L., Yu, K., Zhao, J., Chen, M., Liu, L., Sun, Q., Lin, Z., Zheng, J., Chen, J., & Zhang, J. (2021). STOML2 potentiates metastasis of hepatocellular carcinoma by promoting PINK1-mediated mitophagy and regulates sensitivity to lenvatinib. J Hematol Oncol, 14(1), 16.
27.Ma, W., Chen, Y., Li, W., Xu, Z., Wei, Z., Mou, T., Wu, Z., Cheng, M., Zou, Y., Zhu, Y., Zhou, W., & Geng, Y. (2020). STOML2 Interacts with PHB through Activating MAPK Signaling Pathway to Promote Colorectal Cancer Proliferation. Research Square.
28.Qu, H., Jiang, W., Wang, Y., & Chen, P. (2019). STOML2 as a novel prognostic biomarker modulates cell proliferation, motility and chemo-sensitivity via IL6-Stat3 pathway in head and neck squamous cell carcinoma. Am J Transl, 11(2), 683-695.
29.Xue, B., Dunbrack, R. L., Williams, R. W., Dunker, A. K., & Uversky, V. N. (2010). PONDR-FIT: a meta-predictor of intrinsically disordered amino acids. Biochim Biophys Acta, 1804(4), 996-1010.
30.Li, S., Yu, K., Zhang, Q., Liu, Z., Liu, J., Ju, H.-Q., Zuo, Z., Li, X., Wang, Z., Cheng, H., & Liu, Z.-X. (2021). dSCOPE: a software to detect sequences critical for liquid-liquid phase separation. bioRxiv, 2021.2001.2030.428971.
31.Snyers, L., Umlauf, E., & Prohaska, R. (1998). Oligomeric nature of the integral membrane protein stomatin. J Biol Chem, 273(27), 17221-17226.
32.Salzer, U., Mairhofer, M., & Prohaska, R. (2007). Stomatin A New Paradigm of Membrane Organization Emerges. Dynamic Cell Biology, 13.
33.Rungaldier, S., Umlauf, E., Mairhofer, M., Salzer, U., Thiele, C., & Prohaska, R. (2017). Structure-function analysis of human stomatin: A mutation study. PLoS One, 12(6).
34.Umlauf, E., Csaszar, E., Moertelmaier, M., Schuetz, G. J., Parton, R. G., & Prohaska, R. (2004). Association of stomatin with lipid bodies. J Biol Chem, 279(22), 23699-23709.
35.Mairhofer, M., Steiner, M., Salzer, U., & Prohaska, R. (2009). Stomatin-like protein-1 interacts with stomatin and is targeted to late endosomes. J Biol Chem, 284(42), 29218-29229.
36.Newton, A. C. (1995). Protein kinase C: structure, function, and regulation. J Biol Chem, 270(48), 28495-28498.
37.Hirai, T., & Chida, K. (2003). Protein kinase Czeta (PKCzeta): activation mechanisms and cellular functions. J Biochem, 133(1), 1-7.
38.Xie, Z., Dong, Y., Zhang, M., Cui, M. Z., Cohen, R. A., Riek, U., Neumann, D., Schlattner, U., & Zou, M. H. (2006). Activation of protein kinase C zeta by peroxynitrite regulates LKB1-dependent AMP-activated protein kinase in cultured endothelial cells. J Biol Chem, 281(10), 6366-6375.
39.Chang, M. Y., Huang, D. Y., Ho, F. M., Huang, K. C., & Lin, W. W. (2012). PKC-dependent human monocyte adhesion requires AMPK and Syk activation. PLoS One, 7(7), e40999.
40.Jornayvaz, F. R., & Shulman, G. I. (2010). Regulation of mitochondrial biogenesis. Essays Biochem, 47, 69-84.
41.Willows, R., Sanders, M. J., Xiao, B., Patel, B. R., Martin, S. R., Read, J., Wilson, J. R., Hubbard, J., Gamblin, S. J., & Carling, D. (2017). Phosphorylation of AMPK by upstream kinases is required for activity in mammalian cells. Biochem J, 474(17), 3059-3073.
42.Ghezzi D, Viscomi C, Ferlini A, Gualandi F, Mereghetti P, DeGrandis D, Zeviani M. (2009). Paroxysmal non-kinesigenic dyskinesia is caused by mutations of the MR-1 mitochondrial targeting sequence. Hum Mol Genet. 15;18(6):1058-64.
43.Brydges, S. D., & Carruthers, V. B. (2003). Mutation of an unusual mitochondrial targeting sequence of SODB2 produces multiple targeting fates in Toxoplasma gondii. J Cell Sci, 116(Pt 22), 4675-4685.
44.Lee J, O'Neill RC, Park MW, Gravel M, Braun PE. (2006). Mitochondrial localization of CNP2 is regulated by phosphorylation of the N-terminal targeting signal by PKC: implications of a mitochondrial function for CNP2 in glial and non-glial cells. Mol Cell Neurosci. 31(3):446-62.
45.Owen I, Shewmaker F. (2019) The Role of Post-Translational Modifications in the Phase Transitions of Intrinsically Disordered Proteins. Int J Mol Sci. Nov 5;20(21):5501.
46.楊庭伃。2019。「探討磷酸水解酶PP2A的調節次單元B55δ在調控STOML2磷酸化及活性所扮演的角色」。碩士論文，國立成功大學分子醫學研究所。