Casein Kinase II phosphorylation-induced conformational switch triggers degradation of the papillomavirus E2 protein

The major phosphorylation sites of the bovine papillomavirus E2 transactivator protein are two serine residues, 298 and 301, that are located in a flexible hinge region between the DNA binding and transactivation domains. Phosphorylation of serine residue 301 promotes ubiquitination and rapid degradation of the E2 protein by the proteasome pathway. To understand the mechanism through which phosphorylation regulates the intracellular levels of this unique papillomavirus regulatory protein, we have carried out an extensive mutational analysis of the region surrounding the phosphorylation sites of the E2 protein. Our results indicate that casein kinase II phosphorylates serine 301. However, phosphorylation of serine 301 is not a sufficient recognition motif for proteasomal degradation; other residues that directly surround the phosphorylation sites are crucial for E2 degradation. The phenotypes of E2 proteins mutated in this region indicate that phosphorylation of serine 301 induces a conformational change that leads to degradation of the E2 protein. In support of this model, circular dichroism studies of the conformational tendencies of peptides from this region indicate that phosphorylation at position 301 decreases the local thermodynamic stability of this region. Thus, this region appears to have evolved to display a marginal local thermodynamic stability that can be regulated by phosphorylation, leading to targeted degradation of the E2 protein.     

Cyclin/CDK regulates the nucleocytoplasmic localization of the human papillomavirus E1 DNA helicase

Cyclin-dependent kinases (CDKs) play key roles in eukaryotic DNA replication and cell cycle progression. Phosphorylation of components of the preinitiation complex activates replication and prevents reinitiation. One mechanism is mediated by nuclear export of critical proteins. Human papillomavirus (HPV) DNA replication requires cellular machinery in addition to the viral replicative DNA helicase E1 and origin recognition protein E2. E1 phosphorylation by cyclin/CDK is critical for efficient viral DNA replication. We now show that E1 is phosphorylated by CDKs in vivo and that phosphorylation regulates its nucleocytoplasmic localization. We identified a conserved regulatory region for localization which contains a dominant leucine-rich nuclear export sequence (NES), the previously defined cyclin binding motif, three serine residues that are CDK substrates, and a putative bipartite nuclear localization sequence. We show that E1 is exported from the nucleus by a CRM1-dependent mechanism unless the NES is inactivated by CDK phosphorylation. Replication activities of E1 phosphorylation site mutations are reduced and correlate inversely with their increased cytoplasmic localization. Nuclear localization and replication activities of most of these mutations are enhanced or restored by mutations in the NES. Collectively, our data demonstrate that CDK phosphorylation controls E1 nuclear localization to support viral DNA amplification. Thus, HPV adopts and adapts the cellular regulatory mechanism to complete its reproductive program.     

The death domain of NF-kappa B1 p105 is essential for signal-induced p105 proteolysis

Stimulation of cells with tumor necrosis factor alpha (TNFalpha) triggers NF-kappaB1 p105 proteolysis, releasing associated Rel subunits to translocate into the nucleus and modulate target gene expression. Phosphorylation of serine 927 within the p105 PEST region by the IkappaB kinase (IKK) complex is required to promote p105 proteolysis in response to TNFalpha stimulation. In this study, the role of the p105 death domain (DD) in signal-induced p105 proteolysis is investigated. Endogenous p105 is shown to interact with the IKK complex in HeLa cells, and transient transfection experiments in 293 cells indicate that each of the catalytic components of the IKK complex, IKK1 and IKK2, can bind to p105. Interaction of p105 with both IKK1 and IKK2 is substantially reduced by deletion of the p105 DD or introduction of a specific point mutation (L841A) into the p105 DD homologous to the lpr mutation in Fas. Phosphorylation of immunoprecipitated p105 on serine 927 by purified recombinant IKK1 or IKK2 protein in vitro is dramatically reduced in both DD mutants relative to wild type. Furthermore, both of the DD mutations significantly impair the ability of low concentrations of IKK2 to induce p105 serine 927 phosphorylation and proteolysis in transiently transfected 3T3 cells. However, high levels of transiently expressed IKK2 bypass the requirement for the p105 DD to induce p105 serine 927 phosphorylation. Finally, p105 serine 927 phosphorylation by the endogenous IKK complex after TNFalpha stimulation and subsequent p105 proteolysis is blocked in both p105 DD mutants when stably expressed in HeLa cells. Thus, the p105 DD acts as a docking site for IKK, increasing its local concentration in the vicinity of the p105 PEST region and facilitating efficient serine 927 phosphorylation.     

Critical role of the N-terminal residues of listeriolysin O in phagosomal escape and virulence of Listeria monocytogenes

A putative PEST sequence was recently identified close to the N-terminus of listeriolysin O (LLO), a major virulence factor secreted by the pathogenic Listeria monocytogenes. The deletion of this motif did not affect the secretion and haemolytic activity of LLO, but abolished bacterial virulence. Here, we first tested whether the replacement of the PEST motif of LLO by two different sequences, with either a very high or no PEST score, would affect phagosomal escape, protein stability and, ultimately, the virulence of L. monocytogenes. Then, we constructed LLO mutants with an intact PEST sequence but carrying mutations on either side, or on both sides, of the PEST motif. The properties of these mutants prompted us to construct three LLO mutants carrying single amino acid substitutions in the distal portion of the PEST region (P49A, K50A and P52A; preprotein numbering). Our data demonstrate that the susceptibility of LLO to intracellular proteolytic degradation is not related to the presence of a high PEST score sequence and that the insertion of two residues immediately downstream of the intact PEST sequence is sufficient to impair phagosomal escape and abolish bacterial virulence. Furthermore, we show that single amino acid substitutions in the distal portion of the PEST motif are sufficient to attenuate bacterial -virulence significantly, unravelling the critical role of this region of LLO in the pathogenesis of L. -monocytogenes.     

An assessment of the role of domain F and pest sequences in estrogen receptor half-life and bioactivity

The estrogen receptor (ER) is a rapidly turning over protein, with a half-life of ca. 3–4 h in estrogen target cells. Sequence analysis of the human ER reveals a putative PEST sequence, sequences rich in proline (P), glutamic acid (E), serine (S) and threonine (T), in the carboxy-terminal F domain of the protein. Since PEST sequences have been implicated in the rapid turnover of some proteins, we have used site-directed mutagenesis to investigate the role of the F region containing PEST residues in the stability and bioactivity of the receptor. A truncated form of ER lacking the last 41 amino acids of the protein and encompassing the PEST sequences (amino acids 555 to 567) was made by mutagenesis of the ER cDNA. Pulse-chase experiments, involving immunoprecipitation of [³⁵S]methionine/[³⁵]Scysteine labeled receptors or of receptors covalently labeled with tamoxifen aziridine followed by gel electrophoresis, were used to determine the half-life of the wild-type and truncated ERs. These experiments showed that the turnover rate of the receptors expressed in Chinese hamster ovary and monkey kidney (COS-1) cells was 3 to 5 h and that elimination of the PEST residues did not have a significant effect on the degradation rate of the protein. Moreover, deletion of the last 41 amino acids (F domain) of the ER did not affect transactivation ability, ligand binding affinity, or the phosphorylation pattern of the receptor. Therefore, the role of domain F in ER function remains unclear, but it is not a determinant of the relatively rapid rate of ER turnover in cells.     

Protein phosphatase-1 is a novel regulator of the interaction between IRBIT and the inositol 1,4,5-trisphosphate receptor

IRBIT is an IP3R [IP3 (inositol 1,4,5-trisphosphate) receptor]-binding protein that competes with IP3 for binding to the IP3R. Phosphorylation of IRBIT is essential for the interaction with the IP3R. The unique N-terminal region of IRBIT, residues 1-104 for mouse IRBIT, contains a PEST (Pro-Glu-Ser-Thr) domain with many putative phosphorylation sites. In the present study, we have identified a well-conserved PP1 (protein phosphatase-1)-binding site preceeding this PEST domain which enabled the binding of PP1 to IRBIT both in vitro and in vivo. IRBIT emerged as a mediator of its own dephosphorylation by associated PP1 and, hence, as a novel substrate specifier for PP1. Moreover, IRBIT-associated PP1 specifically dephosphorylated Ser68 of IRBIT. Phosphorylation of Ser68 was required for subsequent phosphorylation of Ser71 and Ser74, but the latter two sites were not targeted by PP1. We found that phosphorylation of Ser71 and Ser74 were sufficient to enable inhibition of IP3 binding to the IP3R by IRBIT. Finally, we have shown that mutational inactivation of the docking site for PP1 on IRBIT increased the affinity of IRBIT for the IP3R. This pinpoints PP1 as a key player in the regulation of IP3R-controlled Ca2+ signals.     

Transferable domain in the G(1) cyclin Cln2 sufficient to switch degradation of Sic1 from the E3 ubiquitin ligase SCF(Cdc4) to SCF(Grr1)

Degradation of Saccharomyces cerevisiae G(1) cyclins Cln1 and Cln2 is mediated by the ubiquitin-proteasome pathway and involves the SCF E3 ubiquitin-ligase complex containing the F-box protein Grr1 (SCF(Grr1)). Here we identify the domain of Cln2 that confers instability and describe the signals in Cln2 that result in binding to Grr1 and rapid degradation. We demonstrate that mutants of Cln2 that lack a cluster of four Cdc28 consensus phosphorylation sites are highly stabilized and fail to interact with Grr1 in vivo. Since one of the phosphorylation sites lies within the Cln2 PEST motif, a sequence rich in proline, aspartate or glutamate, serine, and threonine residues found in many unstable proteins, we fused various Cln2 C-terminal domains containing combinations of the PEST and the phosphoacceptor motifs to stable reporter proteins. We show that fusion of the Cln2 domain to a stabilized form of the cyclin-dependent kinase inhibitor Sic1 (Delta N-Sic1), a substrate of SCF(Cdc4), results in degradation in a phosphorylation-dependent manner. Fusion of Cln2 degradation domains to Delta N-Sic1 switches degradation of Sic1 from SCF(Cdc4) to SCF(Grr1). Delta N-Sic1 fused with a Cln2 domain containing the PEST motif and four phosphorylation sites binds to Grr1 and is unstable and ubiquitinated in vivo. Interestingly, the phosphoacceptor domain of Cln2 binds to Grr1 but is not ubiquitinated and is stable. In summary, we have identified a small transferable domain in Cln2 that can redirect a stabilized SCF(Cdc4) target for SCF(Grr1)-mediated degradation by the ubiquitin-proteasome pathway.     

Mutational analysis of sites in the translational regulator, PHAS-I, that are selectively phosphorylated by mTOR.

Results obtained with PHAS-I proteins having Ser to Ala mutations in the five known phosphorylation sites indicate that mTOR preferentially phosphorylates Thr36 and Thr45. The effects of phosphorylating these sites on eIF4E binding were assessed in a far-Western analysis with a labeled eIF4E probe. Phosphorylation of Thr36 only slightly attenuated binding of PHAS-I to eIF4E, while phosphorylation of Thr45 markedly inhibited binding. Phosphorylation of neither site affected the electrophoretic mobility of the protein, indicating that results of studies that rely solely on a gel-shift assay to assess changes in PHAS-I phosphorylation must be interpreted with caution.     

The N-terminus of Drosophila ESC mediates its phosphorylation and dimerization

The ESC protein, like other Polycomb Group proteins, is required for heritable silencing of the homeotic genes. ESC is phosphorylated in vivo, but the region of ESC that is phosphorylated and its consequences are not known. Here, we show that the amino-terminal region of ESC (residues 1-60) mediates its phosphorylation and dimerization. Phosphorylation of ESC1-60 in vitro by CK1 and CK2 strongly enhances its dimerization. Both phosphorylation and dimerization are conserved in the mammalian ESC homolog EED, suggesting that they play important roles in vivo. One role is suggested by the effect of phosphatase treatment on native ESC complexes, which does not affect the integrity of the 600 kDa ESC/E(Z) complex, but eliminates the 1 MDa ESC/E(Z) complex, which is distinguished from the former by the presence of the additional subunits PCL and RPD3. Thus, stability and perhaps assembly of larger ESC complexes may depend on ESC phosphorylation.     

Intrinsic unstructuredness and abundance of PEST motifs in eukaryotic proteomes

The study of unfolded protein regions has gained importance because of their prevalence and important roles in various cellular functions. These regions have characteristically high net charge and low hydrophobicity. The amino acid sequence determines the intrinsic unstructuredness of a region and, therefore, efforts are ongoing to delineate the sequence motifs, which might contribute to protein disorder. We find that PEST motifs are enriched in the characterized disordered regions as compared with globular ones. Analysis of representative PDB chains revealed very few structures containing PEST sequences and the majority of them lacked regular secondary structure. A proteome-wide study in completely sequenced eukaryotes with predicted unfolded and folded proteins shows that PEST proteins make up a large fraction of unfolded dataset as compared with the folded proteins. Our data also reveal the prevalence of PEST proteins in eukaryotic proteomes (approximately 25%). Functional classification of the PEST-containing proteins shows an over- and under-representation in proteins involved in regulation and metabolism, respectively. Furthermore, our analysis shows that predicted PEST regions do not exhibit any preference to be localized in the C terminals of proteins, as reported earlier.     

Retinoblastoma Protein Contains a C-terminal Motif That Targets It for Phosphorylation by Cyclin-cdk Complexes

Stable association of certain proteins, such as E2F1 and p21, with cyclin-cdk2 complexes is dependent upon a conserved cyclin-cdk2 binding motif that contains the core sequence ZRXL, where Z and X are usually basic. In vitro phosphorylation of the retinoblastoma tumor suppressor protein, pRB, by cyclin A-cdk2 and cyclin E-cdk2 was inhibited by a short peptide spanning the cyclin-cdk2 binding motif present in E2F1. Examination of the pRB C terminus revealed that it contained sequence elements related to ZRXL. Site-directed mutagenesis of one of these sequences, beginning at residue 870, impaired the phosphorylation of pRB in vitro. A synthetic peptide spanning this sequence also inhibited the phosphorylation of pRB in vitro. pRB C-terminal truncation mutants lacking this sequence were hypophosphorylated in vitro and in vivo despite the presence of intact cyclin-cdk phosphoacceptor sites. Phosphorylation of such mutants was restored by fusion to the ZRXL-like motif derived from pRB or to the ZRXL motifs from E2F1 or p21. Phospho-site-specific antibodies revealed that certain phosphoacceptor sites strictly required a C-terminal ZRXL motif whereas at least one site did not. Furthermore, this residual phosphorylation was sufficient to inactivate pRB in vivo, implying that there are additional mechanisms for directing cyclin-cdk complexes to pRB. Thus, the C terminus of pRB contains a cyclin-cdk interaction motif of the type found in E2F1 and p21 that enables it to be recognized and phosphorylated by cyclin-cdk complexes.     

Phosphomimetic mutations enhance oligomerization of phospholemman and modulate its interaction with the Na/K-ATPase

Na/K-ATPase (NKA) activity is dynamically regulated by an inhibitory interaction with a small transmembrane protein, phospholemman (PLM). Inhibition is relieved upon PLM phosphorylation. Phosphorylation may alter how PLM interacts with NKA and/or itself, but details of these interactions are unknown. To address this, we quantified FRET between PLM and its regulatory target NKA in live cells. Phosphorylation of PLM was mimicked by mutation S63E (PKC site), S68E (PKA/PKC site), or S63E/S68E. The dependence of FRET on protein expression in live cells yielded information about the structure and binding affinity of the PLM-NKA regulatory complex. PLM phosphomimetic mutations altered the quaternary structure of the regulatory complex and reduced the apparent affinity of the PLM-NKA interaction. The latter effect was likely due to increased oligomerization of PLM phosphomimetic mutants, as suggested by PLM-PLM FRET measurements. Distance constraints obtained by FRET suggest that phosphomimetic mutations slightly alter the oligomer quaternary conformation. Photon-counting histogram measurements revealed that the major PLM oligomeric species is a tetramer. We conclude that phosphorylation of PLM increases its oligomerization into tetramers, decreases its binding to NKA, and alters the structures of both the tetramer and NKA regulatory complex.     

The N-terminal module of HPV16 E7 is an intrinsically disordered domain that confers conformational and recognition plasticity to the oncoprotein

The HPV16 E7 oncoprotein is an extended dimer, with a stable and cooperative fold, but that displays properties of "natively unfolded" proteins. Two regions of conserved sequence are found in E7 proteins, where the N-terminus (1-40) includes the retinoblastoma tumor suppressor binding and casein kinase II phosphorylation sites. A fragment containing the highly acidic N-terminal half shows an apparently disordered conformation by far-UV-circular dichroism (CD) at neutral pH, and its hydrodynamic radius is much larger than a neutral peptide of the same length. Trifluoroethanol and micellar concentrations of sodium dodecyl sulfate stabilize a much more helical structure at pH 4.0 than at pH 7.5, while submicellar concentrations of the detergent yield a beta-strand. The shape, pH, and temperature dependence of the CD spectrum at pH 7.5 are indicative of a poly proline type II structure. This structure is stabilized by phosphorylation, which would translate into increased transforming activity in the cell. Thus, the intrinsically disordered properties of the N-terminal module of E7 are responsible for the structural plasticity of the oncoprotein. Although the domain is not a compact and cooperatively folded unit, it is a bona fide functional domain, evolved to maintain a dynamic but extended structure in the cell. These properties allow adaptation to a variety of protein targets and expose the PEST degradation sequence that regulates its turnover in the cell, a modification of which leads to the accumulation of E7 species with consequences in the transformation process.     

beta-Amyloid precursor is a PEST protein

The beta-amyloid peptide is generated by proteolytic processing of a family of beta-amyloid precursor proteins. Here we report that beta-amyloid precursor proteins have a primary structure motif known as a PEST sequence, which is predictive of the class of most protease-sensitive rapidly turning over proteins. Consistent with this, the precursors were extraordinarily susceptible to degradation by the calcium-dependent protease calpain I. The identification of beta-amyloid precursors as PEST sequence-containing proteins has implications for both the normal cellular function of beta-amyloid precursor proteins and the mechanisms regulating their expression and processing.