Investigate the Role of the B55δ Regulatory Subunit of Protein Phosphatase 2A in Regulating Phosphorylation and Activities of STOML2

Abstract

Protein phosphatase 2A (PP2A) is one of the major serine/threonine phosphatases and widely expressed in eukaryotic cells A PP2A holoenzyme consists of three subunits including a scaffolding A subunit a catalytic C subunit and a variable regulatory B subunit which determines the substrate specificity and subcellular localization of PP2A The B regulatory subunit has been classified into four subfamilies B (B55/PR55) B’ (B56/PR61) B’’ (PR72/PR130) and B’’’ (PR93/SG2NA) The current study focused on B55δ which belongs to the B (B55/PR55) family In addition to playing a key role in regulating entry and exit of mitosis B55δ was recently shown to play a negative role in regulating survival and proliferation of tumor-infiltrating T cells STOML2 also known as SLP-2 and paratarg-7 is a mitochondrial protein and regulates the biogenesis and activities of mitochondria Expression of STOML2 is upregulated in a variety of cancer and promotes tumorigenesis migration and invasion of tumor cells Additionally STOML2 plays an important role in regulating T cell receptor signaling and T cell activation The PP2A-B55δ holoenzyme was shown to dephosphorylate STOML2 and the dysregulated interaction between PP2A-B55δ and STOML2 is one of the hallmarks of plasma cell diseases Importantly the functional role of PP2A-B55δ in regulating reversible phosphorylation of STOML2 is largely unknown We firstly investigated the role of B55δ in regulating the protein and phosphorylation level of STOML2 In HeLa cells overexpression or knockdown of B55δ had no significant impact on levels of STOML2 at steady state Upon serum stimulation the protein levels of STOML2 still remained unaffected by B55δ Transiently transfection of B55δ or B55δ-specific shRNA also did not alter the STOML2 protein expression in HeLa cells In addition the protein expression of STOML2 was not affected in Jurkat cells with stable knockdown of B55δ expression Results of Phos-tagTM SDS-PAGE analysis showed that knockdown of B55δ increased phosphorylation of STOML2 At steady state at least three phosphorylated forms of STOML2 were identified and levels of the phosphorylated form of STOML2 were significantly decreased by both B55δ and B56γ3 Furthermore under co-expression of PKC ζ and PMA treatment we identified two phosphorylated bands of STOML2 catalyzed by PKC ζ by Phos-tagTM SDS-PAGE analysis The levels of one of the phosphorylated bands of STOML2 were more selectively decreased by B55δ than by B56γ3 To investigate whether the biological function of STOML2 on mitochondria is regulated by B55δ we investigated the mitochondrial biogenesis regulated by B55δ using mitochondrial staining and fluorescence microscopy The integrated density fluorescence analysis showed that transient STOML2 overexpression increased mitochondrial staining and both exogenous B55δ and B56γ3 reduced the enhancement of mitochondrial staining by STOML2 Furthermore we investigated whether the mitochondrial distribution of STOML2 is regulated by B55δ and we found that endogenous STOML2 exhibits punctate distribution while exogenous STOML2 formed cytoplasmic aggregates when B55δ or B56γ3 was overexpressed By colocalization analysis transient B55δ overexpression slightly affect mitochondrial localization of endogenous STOML2 but both B55δ and B56γ3 significantly impaired mitochondrial distribution of exogenous STOML2 In summary our results demonstrated that overexpression of B55δ had no effect on the protein level of STOML2 both at steady state and under serum stimulation in both HeLa and Jurkat cells However knockdown of B55δ expression also showed no effect on the protein level of STOML2 in both HeLa and Jurkat cells Although both B55δ and B56γ3 reduced the level of certain phosphorylated forms of STOML2 B55δ selectively decreased levels of phosphorylated STOML2 catalyzed by PKC ζ activation compared to B56γ3 Interestingly both B55δ and B56γ3 decreased STOML2-enhanced mitochondrial biogenesis and impaired mitochondrial localization of exogenous STOML2

Date of Award	2019
Original language	English
Supervisor	Chi-Wu Chiang (Supervisor)