PI(4,5)P2 is phosphorylated by PI3K to produce PI(3,4,5)P3, which promotes membrane localization of PDK1, which then phosphorylates and activates AKT. an insertion mutant of GRP78 at its N-terminus domain name, while retaining stable expression and the ability to translocate to the cell surface as the wild-type protein, exhibited reduced complex formation with p85 and production of PIP3. Thus, our studies provide a mechanistic explanation for the regulation of the PI3K/AKT signaling by sGRP78. Our findings suggest that targeting sGRP78 may suppress therapeutic Glycerol phenylbutyrate resistance in cancer cells and offer a novel strategy to suppress PI3K activity. Introduction The 78 kDa glucose-regulated protein (GRP78), also referred to as BiP/HSPA5, is usually a major endoplasmic reticulum (ER) chaperone with anti-apoptotic properties [1] and a grasp regulator of ER stress signaling [2], [3]. Tumor cells are subjected to ER stress due to intrinsic factors of altered metabolism and extrinsic factors of hypoxia and nutrient deprivation. ER stress induction of GRP78 in cancer cells favors cell survival, tumor progression [4], [5] and confers drug resistance in both proliferating and dormant cancer cells, as well as tumor associated endothelial cells [6]C[11]. Therefore, understanding how GRP78 exerts its pleiotrophic effects on cell proliferation and survival is usually of major importance. Traditionally GRP78 has been regarded as an ER lumenal protein due to its carboxyl KDEL retention motif [12]. Recently, a subfraction of GRP78 was found to localize to the surface of specific cell types, particularly in cancer cells [13]C[16]. Cell surface proteome profiling of tumor cells revealed a relative abundance of heat shock chaperones and glucose-regulated proteins, including GRP78 [17]. Importantly, preferential expression of GRP78 on the surface of tumor cells but not in normal organs enables specific tumor targeting, leading to tumor suppression without harmful effects on normal tissues [18]C[21]. Evidence is usually emerging that sGRP78 can form complexes with specific cell surface proteins and regulate signal transduction [13], [14], [16], such as being a co-receptor for the proteinase inhibitor 2-macroglobulin (2-M*) induced signal transduction for cancer survival and metastasis [22], Glycerol phenylbutyrate [23]. Cripto, a GPI-anchored cell surface protein key to human tumor progression, and sGRP78 Glycerol phenylbutyrate form a complex and collaborate to inhibit TGF- signaling and enhance cell growth and PI3K/AKT activation [24], [25]. Additionally, sGRP78 is required for T-cadherin-dependent endothelial cell survival [26], activation of apoptosis mediated Mouse monoclonal to CRTC3 by Kringle 5 [27], [28] and extracellular Par-4 and TRAIL [29], as well as viral entry into host cells [30], [31]. Recently we exhibited cell surface localization of GRP78 is usually regulated by ER retrieval machinery and enhanced by depletion of Ca2+ from the ER [32]. Cancer cells are often subjected to ER stress, which are aggravated by cytotoxic therapy Glycerol phenylbutyrate leading to resistance. However, whether pathological stress, such as development of therapeutic resistance, leads to relocalization of GRP78 to the cell surface is not known. The PI3K/AKT pathway is usually activated in a wide array of cancers leading to proliferation and therapeutic resistance [33]. The PI3K has two subunits, the p85 regulatory subunit and the p110 catalytic subunit. For PI3K activation, tyrosine phosphorylation of the p85 regulatory subunit of PI3K relieves its inhibitory activity on PI3K, leading to its activation. Upon binding to the activated growth factor receptor, PI3K is usually recruited to the plasma membrane. PI(4,5)P2 is usually Glycerol phenylbutyrate phosphorylated by PI3K to yield PI(3,4,5)P3, which promotes membrane localization of PDK1, which then phosphorylates and activates AKT. Through knockdown of GRP78 by siRNA, ligation of cell surface GRP78 with antibody and in genetic models of cancer, GRP78 has been established as a novel regulator of PI3K signaling both in vitro and in vivo [16], [25], [34], [35]. While there can be multiple mechanisms whereby GRP78 can affect AKT activation, it has been reported that antibody targeting the N-terminus of.