Ribosome-binding protein p34 is a member of the leucine-rich-repeat-protein superfamily

With the aim of identifying new intracellular binding partners for acidic fibroblast growth factor (aFGF), proteins from U2OS human osteosarcoma cells were adsorbed to immobilized aFGF. One of the adsorbed proteins is a member of the leucine-rich repeat protein family termed ribosome-binding protein p34 (p34). This protein has previously been localized to endoplasmic reticulum membranes and is thought to span the membrane with the N terminus on the cytosolic side. Confocal microscopy of cells transfected with Myc-p34 confirmed the endoplasmic reticulum localization, and Northern blotting determined p34 mRNA to be present in a multitude of different tissues. Cross-linking experiments indicated that the protein is present in the cell as a dimer. In vitro translated p34 was found to interact with maltose-binding protein-aFGF through its cytosolic coiled-coil domain. The interaction between aFGF and p34 was further characterized by surface plasmon resonance, giving a K D of 1.4 ؎ 0.3 M. Even though p34 interacted with mitogenic aFGF, it bound poorly to the non-mitogenic aFGF(K132E) mutant, indicating a possible involvement of p34 in intracellular signaling by aFGF.

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confidence: 82%

Section: Identification Of P34 As a Protein Thatmentioning

confidence: 99%

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Identification of Ribosome-binding Protein p34 as an Intracellular Protein That Binds Acidic Fibroblast Growth Factor

Skjerpen¹,

Wesche²,

Olsnes

2002

“…Suppression of transformation and IN-HAT activity map to amino acids 150 -174, slightly N-terminal to the acidic region. LRRs generally mediate protein-protein interactions (39), and the LRR of pp32 mediates its nucleocytoplasmic shuttling via binding to CRM1 (34). The Rb-binding region of pp32 was mapped using V5 epitope-tagged constructs lacking the acidic region, the LRR, or both (Fig.…”

Section: Resultsmentioning

confidence: 99%

Phosphorylated Retinoblastoma Protein Complexes with pp32 and Inhibits pp32-mediated Apoptosis

Adegbola¹,

Pasternack

2005

The retinoblastoma gene product (Rb) is a tumor suppressor that affects apoptosis paradoxically. Most sporadic cancers inactivate Rb by preferentially targeting the pathway that regulates Rb phosphorylation, resulting in resistance to apoptosis; this contrasts with Rb inactivation by mutation, which is associated with high rates of apoptosis. How phosphorylated Rb protects cells from apoptosis is not well understood, but there is evidence that Rb may sequester a pro-apoptotic nuclear factor. pp32 (ANP32A) is a pro-apoptotic nuclear phosphoprotein, the expression of which is commonly increased in cancer. We report that hyperphosphorylated Rb interacts with pp32 but not with the closely related proteins pp32r1 and pp32r2. We further demonstrate that pp32-Rb interaction inhibits the apoptotic activity of pp32 and stimulates proliferation. These results suggest a mechanism whereby cancer cells gain both a proliferative and survival advantage when Rb is inactivated by hyperphosphorylation.The retinoblastoma protein (Rb) 1 is a nuclear phosphoprotein that regulates proliferation, differentiation, and apoptosis. As a tumor suppressor, Rb inhibits proliferation by repressing E2F1-mediated transcription when hypophosphorylated. Hyperphosphorylation of Rb relieves E2F1 repression and allows cell cycle progression to occur (1). The importance of Rb is underscored by the fact that Rb function is disrupted in virtually all human cancers (2). Paradoxically and inconsistent with its role as a tumor suppressor, hyperphosphorylated wild-type Rb inhibits apoptosis in both cell culture and animal models (3)(4)(5)(6)(7)(8)(9)(10). Because Rb inactivation is pivotal for carcinogenesis, this poses the problem of how cancer cells escape apoptosis when Rb function is disrupted.Inherited cancers and cells in which Rb is inactivated by mutation have increased rates of both proliferation and apoptosis (11). Most sporadic cancers preferentially inactivate Rb by hyperphosphorylation, which may occur through mutation of cyclin D, cdk4, or p16. Such cancers are generally slow growing and resistant to apoptosis induced by chemotherapy or radiation (12). It is possible that in these cancers the tumor suppressor function of Rb is inhibited, whereas the anti-apoptotic function remains intact (13). Inactivation by hyperphosphorylation might promote proliferation by increasing free E2F1, as well as inhibit apoptosis by retaining the anti-apoptotic function of Rb. This is consistent with evidence suggesting that it is the hyperphosphorylated form of Rb rather than Rb per se that protects cells from apoptosis (14). The induction of apoptosis in various cell lines is accompanied by a shift in Rb from the hyperphosphorylated to the hypophosphorylated form (15,16). Rb dephosphorylation, which has been shown to be required for apoptosis, occurs in the early stage of apoptosis (17,18). Inhibition of Rb dephosphorylation prevents apoptosis, whereas induction of dephosphorylation leads to apoptosis (19). In DBA/2 mice, increased levels of hyperphosphoryla...

“…Most recently, the primary sequence of p34 was deduced, showing the protein to be a type II membrane protein with a large cytosolic domain. It contains 4.5 tandem leucine-rich repeats (23-24 amino acids in length) and an abundance of charged amino acids (8). p180 was first identified as a soluble proteolytic fragment that inhibited ribosome binding to intact microsomes (5).…”

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confidence: 99%

Receptor-mediated Ribosome Binding to Liposomes Depends on Lipid Composition

Savitz¹,

Meyer²

1997

Ribosome binding to the endoplasmic reticulum has been traditionally studied using an in vitro assay in which potential ribosome receptors have been purified, incorporated into synthetic liposomes, and tested for activity. One such receptor (180 kDa; "p180") has been shown to bind ribosomes with high affinity in such a system when purified to homogeneity. This result has been challenged by data generated in other laboratories, and as a result, doubt has lingered as to the authenticity of p180 as a ribosome receptor. The contribution of the major difference between these studies, the lipid composition of the liposomes used in the in vitro assays, was assessed when identical fractions of rough endoplasmic reticulum-specific membrane proteins were incorporated into liposomes composed of only phosphatidylcholine (as used in other laboratories), a 50:50 mix of phosphatidylcholine and phosphatidylserine (as used in our original studies), or lipids derived from canine pancreatic microsomes (as a physiologically relevant control). The presence of PS was found to be crucial for the incorporation into and ribosome binding activity of p180 in liposomes. These observations are compatible with published studies on the importance of acidic phospholipids in ribosome binding to intact microsomes and reconcile the apparently conflicting in vitro results surrounding the assignment of p180 as a ribosome receptor.