Key Spec Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, M||WB||M||Culture Supernatant||Monoclonal Antibody|
|Description||Anti-Alpha4 Antibody, clone 5F6|
|Application||This Anti-Alpha4 Antibody, clone 5F6 is validated for use in WB for the detection of Alpha4.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||2 years at -20°C from date of shipment|
|Material Size||200 µL|
|Anti-Alpha4, clone 5F6 - 30472||30472|
|Anti-Alpha4, clone 5F6 -2607877||2607877|
|Anti-Alpha4, clone 5F6 -2767032||2767032|
|Anti-Alpha4, clone 5F6 -2796824||2796824|
|Reference overview||Pub Med ID|
|Phosphorylation of eIF2α triggered by mTORC1 inhibition and PP6C activation is required for autophagy and is aberrant in PP6C-mutated melanoma.|
Wengrod, J; Wang, D; Weiss, S; Zhong, H; Osman, I; Gardner, LB
Science signaling 8 ra27 2015
Amino acid deprivation promotes the inhibition of the kinase complex mTORC1 (mammalian target of rapamycin complex 1) and activation of the kinase GCN2 (general control nonrepressed 2). Signaling pathways downstream of both kinases have been thought to independently induce autophagy. We showed that these two amino acid-sensing systems are linked. We showed that pharmacological inhibition of mTORC1 led to activation of GCN2 and phosphorylation of the eukaryotic initiation factor 2α (eIF2α) in a mechanism dependent on the catalytic subunit of protein phosphatase 6 (PP6C). Autophagy induced by pharmacological inhibition of mTORC1 required PP6C, GCN2, and eIF2α phosphorylation. Although some of the PP6C mutants found in melanoma did not form a strong complex with PP6 regulatory subunits and were rapidly degraded, these mutants paradoxically stabilized PP6C encoded by the wild-type allele and increased eIF2α phosphorylation. Furthermore, these PP6C mutations were associated with increased autophagy in vitro and in human melanoma samples. Thus, these data showed that GCN2 activation and phosphorylation of eIF2α in response to mTORC1 inhibition are necessary for autophagy. Additionally, we described a role for PP6C in this process and provided a mechanism for PP6C mutations associated with melanoma.
|Regulation of autophagy by coordinated action of mTORC1 and protein phosphatase 2A.|
Wong, PM; Feng, Y; Wang, J; Shi, R; Jiang, X
Nature communications 6 8048 2015
Autophagy is a cellular catabolic process critical for cell viability and homoeostasis. Inhibition of mammalian target of rapamycin (mTOR) complex-1 (mTORC1) activates autophagy. A puzzling observation is that amino acid starvation triggers more rapid autophagy than pharmacological inhibition of mTORC1, although they both block mTORC1 activity with similar kinetics. Here we find that in addition to mTORC1 inactivation, starvation also causes an increase in phosphatase activity towards ULK1, an mTORC1 substrate whose dephosphorylation is required for autophagy induction. We identify the starvation-stimulated phosphatase for ULK1 as the PP2A-B55α complex. Treatment of cells with starvation but not mTORC1 inhibitors triggers dissociation of PP2A from its inhibitor Alpha4. Furthermore, pancreatic ductal adenocarcinoma cells, whose growth depends on high basal autophagy, possess stronger basal phosphatase activity towards ULK1 and require ULK1 for sustained anchorage-independent growth. Taken together, concurrent mTORC1 inactivation and PP2A-B55α stimulation fuel ULK1-dependent autophagy.
|Mig12, a novel Opitz syndrome gene product partner, is expressed in the embryonic ventral midline and co-operates with Mid1 to bundle and stabilize microtubules.|
Berti, Caterina, et al.
BMC Cell Biol., 5: 9 (2004) 2004
|The PP2A-associated protein alpha4 is an essential inhibitor of apoptosis.|
Kong, Mei, et al.
Science, 306: 695-8 (2004) 2004
Despite evidence that protein kinases are regulators of apoptosis, a specific role for phosphatases in regulating cell survival has not been established. Here we show that alpha4, a noncatalytic subunit of protein phosphatase 2A (PP2A), is required to repress apoptosis in murine cells. alpha4 is a nonredundant regulator of the dephosphorylation of the transcription factors c-Jun and p53. As a result of alpha4 deletion, multiple proapoptotic genes were transcribed. Either inhibition of new protein synthesis or Bcl-xL overexpression suppressed apoptosis initiated by alpha4 deletion. Thus, mammalian cell viability depends on repression of transcription-initiated apoptosis mediated by a component of PP2A.
|Parallel purification of three catalytic subunits of the protein serine/threonine phosphatase 2A family (PP2A(C), PP4(C), and PP6(C)) and analysis of the interaction of PP2A(C) with alpha4 protein.|
Kloeker, Susanne, et al.
Protein Expr. Purif., 31: 19-33 (2003) 2003
The protein serine/threonine phosphatase (PP) type 2A family consists of three members: PP2A, PP4, and PP6. Specific rabbit and sheep antibodies corresponding to each catalytic subunit, as well as a rabbit antibody recognizing all three subunits, were utilized to examine the expression of these enzymes in select rat tissue extracts. PP2A, PP4, and PP6 catalytic subunits (PP2A(C), PP4(C), and PP6(C), respectively) were detected in all rat tissue extracts examined and exhibited some differences in their levels of expression. The expression of alpha4, an interacting protein for PP2A family members that may function downstream of the target of rapamycin (Tor), was also examined using specific alpha4 sheep antibodies. Like the phosphatase catalytic subunits, alpha4 was ubiquitously expressed with particularly high levels in the brain and thymus. All three PP2A family members, but not alpha4, bound to the phosphatase affinity resin microcystin-Sepharose. The phosphatase catalytic subunits were purified to apparent homogeneity (PP2A(C) and PP4(C)) or near homogeneity (PP6(C)) from bovine testes soluble extracts following ethanol precipitation and protein extraction. In contrast to PP2A(C), PP4(C) and PP6(C) exhibited relatively low phosphatase activity towards several substrates. Purified PP2A(C) and native PP2A in cellular extracts bound to GST-alpha4, and co-immunoprecipitated with endogenous alpha4 and ectopically expressed myc-tagged alpha4. The interaction of PP2A(C) with alpha4 was unaffected by rapamycin treatment of mammalian cells; however, protein serine/threonine phosphatase inhibitors such as okadaic acid and microcystin-LR disrupted the alpha4/PP2A complex. Together, these findings increase our understanding of the biochemistry of alpha4/phosphatase complexes and suggest that the alpha4 binding site within PP2A may include the phosphatase catalytic domain.
|Developmental expression of alpha4 protein phosphatase regulatory subunit in tissues affected by Opitz syndrome.|
Everett, Allen D and Brautigan, David L
Dev. Dyn., 224: 461-4 (2002) 2002
Mutations in the Mid1 gene are responsible for X-linked Opitz syndrome, characterized by midline defects of the brain, face, heart, and trachea. The regulatory subunit alpha4 binds protein phosphatase 2A to Mid1 and promotes Mid1 dephosphorylation, in opposition to MAP kinase. To examine the relationship between alpha4 expression and human defects in Opitz syndrome, developmental expression of alpha4 in embryonic day (E) 8-E16 mouse embryos was mapped by immunohistochemistry. At E10, alpha4 was first detected only in the heart. At E11, alpha4 was expressed in epithelium of the mandibular and maxillary arches and in specific subsets of mesenchymal cells within the arches. The fetal heart and brain also highly expressed alpha4. At E16, alpha4 expression broadened to include the heart, brain, skeletal muscle, and tracheal and esophageal epithelium but not smooth muscle. Consistent with immunolocalization in embryos, Western immunoblotting of adult rabbit tissues demonstrated high levels of alpha4 expression in brain, heart, lung, liver, and skeletal muscle with low expression in kidney, uterus, and intestine. Expression in slow type I skeletal muscle was much higher than in fast type II muscle. By using double immunohistochemical staining, alpha4 was coexpressed in mouse skeletal muscle cells containing slow type myosin. Expression of alpha4 overlaps with the tissue defects in Opitz syndrome. The alpha4 gene lies in the linkage interval for FG syndrome, characterized by skeletal muscle and brain defects that coincide closely to the expression pattern of alpha4.
|Phosphorylation and microtubule association of the Opitz syndrome protein mid-1 is regulated by protein phosphatase 2A via binding to the regulatory subunit alpha 4.|
Liu, J, et al.
Proc. Natl. Acad. Sci. U.S.A., 98: 6650-5 (2001) 2001
Opitz syndrome (OS) is a human genetic disease characterized by deformities such as cleft palate that are attributable to defects in embryonic development at the midline. Gene mapping has identified OS mutations within a protein called Mid1. Wild-type Mid1 predominantly colocalizes with microtubules, in contrast to mutant versions of Mid1 that appear clustered in the cytosol. Using yeast two-hybrid screening, we found that the alpha4-subunit of protein phosphatases 2A/4/6 binds Mid1. Epitope-tagged alpha4 coimmunoprecipitated endogenous or coexpressed Mid1 from COS7 cells, and this required only the conserved C-terminal region of alpha4. Localization of Mid1 and alpha4 was influenced by one another in transiently transfected cells. Mid1 could recruit alpha4 onto microtubules, and high levels of alpha4 could displace Mid1 into the cytosol. Metabolic (32)P labeling of cells showed that Mid1 is a phosphoprotein, and coexpression of full-length alpha4 decreased Mid1 phosphorylation, indicative of a functional interaction. Association of green fluorescent protein-Mid1 with microtubules in living cells was perturbed by inhibitors of MAP kinase activation. The conclusion is that Mid1 association with microtubules, which seems important for normal midline development, is regulated by dynamic phosphorylation involving MAP kinase and protein phosphatase that is targeted specifically to Mid1 by alpha4. Human birth defects may result from environmental or genetic disruption of this regulatory cycle.
|B cell receptor-associated protein alpha4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A.|
Murata, K, et al.
Proc. Natl. Acad. Sci. U.S.A., 94: 10624-9 (1997) 1997
Recently, TAP42 was isolated as a high copy suppressor of sit4-, a yeast phosphatase related to protein phosphatase 2A (PP2A). TAP42 is related to the murine alpha4 protein, which was discovered independently by its association with Ig-alpha in the B cell receptor complex. Herein we show that a glutathione S-transferase (GST)-alpha4 fusion protein bound the catalytic subunit (C) of human PP2A from monomeric or multimeric preparations of PP2A in a "pull-down" assay. In an overlay assay, the GST-alpha4 protein bound to the phosphorylated and unphosphorylated forms of C that were separated in two-dimensional gels and immobilized on filters. The results show direct and exclusive binding of alpha4 to C. This is unusual because all known regulatory B subunits, or tumor virus antigens, bind stably only to the AC dimer of PP2A. The alpha4-C form of PP2A had an increased activity ratio compared with the AC form of PP2A when myelin basic protein phosphorylated by mitogen-activated protein kinase and phosphorylase a were used as substrates. Recombinant alpha4 cleaved from GST was phosphorylated by p56(lck) tyrosine kinase and protein kinase C. A FLAG-tagged alpha4 expressed in COS7 cells was recovered as a protein containing phosphoserine and coimmunoprecipitated with the C but not the A subunit of PP2A. Treatment of cells with rapamycin prevented the association of PP2A with FLAG-alpha4. The results reveal a novel heterodimer alpha4-C form of PP2A that may be involved in rapamycin-sensitive signaling pathways in mammalian cells.