Investigate the cellular functions of type 2A-interacting protein (TIP)

摘要

Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase involved in regulating a variety of cellular activities in mammalian cells The canonical PP2A complex consists of a heterodimeric core enzyme including a scaffolding subunit (A) and a catalytic subunit (PP2Ac) and a variable regulatory subunit (B) In addition to the canonical PP2A complex several molecules such as ?4 and type 2A-interacting protein (TIP) have been identified to associate with PP2Ac to form non-canonical PP2A complexes To understand more about the roles of TIP in cellular functions we established pools of NIH3T3 HeLa Hep3B and HepG2 cells with stable TIP overexpression or knockdown of TIP and characterized these cells according to their morphological characteristics growth properties motility and migration We found that TIP overexpression increased proliferation of NIH3T3 cells and HepG2 cells whereas TIP overexpression reduced proliferation of HeLa cells HeLa cells with stable TIP knockdown showed increased numbers of cells with a spindle-shaped morphology Consistent with the morphology change TIP overexpression reduced cell motility and wound-healing migration in HeLa cells whereas HeLa cells with stable TIP knockdown showed enhanced motility and migration Concomitantly TIP overexpression increased staining of actin web while TIP knockdown increased amounts of actin stress fibers Next we investigated signaling pathways regulated by TIP TIP overexpression slightly down-regulated the levels of the B55 family regulatory subunits of PP2A whereas TIP overexpression increases protein levels of B56γ regulatory subunits ?4 and Akt in HeLa cells Knockdown of TIP by siRNA reduced levels of B56γ2 B56γ3 ?4 and Akt in HeLa cells TIP overexpression increased protein levels of ?4 and Akt but not B56γ in HepG2 cells Conversely modulating TIP levels did not change the levels of the A and C subunits In addition TIP overexpression increased phosphorylation levels of Akt at S473 whereas TIP overexpression decreased phosphorylation levels of Akt at T308 and phosphorylation levels of P70S6K at T389 TIP appears to affect P70S6K phosphorylation independent of mTORC1 since TIP overexpression did not affect phosphorylation levels of 4EBP1 at S65 In summary our data demonstrate that TIP can either up-regulate or down-regulate cell proliferation depending on cell types TIP regulates morphology cell motility and migration through modulating actin filament rearrangements Taken together TIP-regulated cellular phenotypes may be attributable to modulating signaling pathways involving PP2A B55 family subunits B56γ family subunits Akt ?4 and P70S6K

獎項日期	2014 9月 3
原文	English
監督員	Chi-Wu Chiang (Supervisor)