Human Fas-associated factor 1, interacting with ubiquitinated proteins and valosin-containing protein, is involved in the ubiquitin-proteasome pathway

Human Fas-associated factor 1 (hFAF1) is a novel protein having multiubiquitin-related domains. We investigated the cellular functions of hFAF1 and found that valosin-containing protein (VCP), the multiubiquitin chain-targeting factor in the degradation of the ubiquitin-proteasome pathway, is a binding partner of hFAF1. hFAF1 is associated with the ubiquitinated proteins via the newly identified N-terminal UBA domain and with VCP via the C-terminal UBX domain. The overexpression of hFAF1 and a truncated UBA domain inhibited the degradation of ubiquitinated proteins and increased cell death. These results suggest that hFAF1 binding to ubiquitinated protein and VCP is involved in the ubiquitin-proteasome pathway. We hypothesize that hFAF1 may serve as a scaffolding protein that regulates protein degradation in the ubiquitin-proteasome pathway.     

Tyrosine phosphorylation of ATPase p97 regulates its activity during ERAD

In eukaryotic cells, the endoplasmic reticulum-associated degradation (ERAD) pathway is essential for the disposal of misfolded proteins. Recently, we demonstrated the existence of a higher order complex consisting of the ER bound E3 ligase gp78, p97, PNGase, and HR23B in mammals. This complex may serve to facilitate the routing of misfolded glycoproteins out of the ER to the cytosol where they are degraded by the proteasome. In this complex, p97 functions as an organizer to mediate the interactions with gp78 and the deglycosylating enzyme PNGase. A novel protein-binding motif of mouse p97 was identified that consists of its last 10 amino acid residues; this motif is sufficient to mediate the interaction of p97 with PNGase and Ufd3. Phosphorylation of p97’s highly conserved penultimate tyrosine residue, completely blocks binding of both PNGase and Ufd3 to mp97. We have found that c-Src kinase directly and selectively phosphorylated the penultimate tyrosine of p97 in vitro, and that overexpression of c-Src significantly increased the phosphorylation level of p97 in cells and caused accumulation of the ERAD substrate TCRα-GFP, as well as ubiquitin-conjugated substrates. These results suggest a role for p97 phosphorylation in the degradation of misfolded glycoproteins.     

Serine 403 phosphorylation of p62/SQSTM1 regulates selective autophagic clearance of ubiquitinated proteins

Selective macroautophagy (autophagy) of ubiquitinated protein is implicated as a compensatory mechanism of the ubiquitin-proteasome system. p62/SQSTM1 is a key molecule managing autophagic clearance of polyubiquitinated proteins. However, little is known about mechanisms controlling autophagic degradation of polyubiquitinated proteins. Here, we show that the specific phosphorylation of p62 at serine 403 (S403) in its ubiquitin-associated (UBA) domain increases the affinity between UBA and polyubiquitin chain, resulting in efficiently targeting polyubiquitinated proteins in "sequestosomes" and stabilizing sequestosome structure as a cargo of ubiquitinated proteins for autophagosome entry. Casein kinase 2 (CK2) phosphorylates S403 of p62 directly. Furthermore, CK2 overexpression or phosphatase inhibition reduces the formation of inclusion bodies of the polyglutamine-expanded huntingtin exon1 fragment in a p62-dependent manner. We propose that phosphorylation of p62 at S403 regulates autophagic clearance of ubiquitinated proteins and protein aggregates that are poorly degraded by proteasomes.     

Characterization of the aggregation-prevention activity of p97/valosin-containing protein

The 97 kDa valosin-containing protein (VCP) belongs to a highly conserved AAA (ATPases associated with a variety of activities) family and contains two ATPase domains, D1 and D2. VCP participates in numerous cellular activities, such as membrane fusion, postmitotic Golgi reassembly, endoplasmic reticulum-associated degradation, ubiquitin-proteasome-mediated proteolysis, and many others. In performing these activities, VCP presumably acts as a molecular chaperone that prevents protein aggregation and modifies protein conformation. In this study, we characterized the aggregation-prevention activity of VCP and identified the structural requirement for this activity. We used multiple methods to treat aggregation-prone luciferase (Luc) and showed that VCP prevents the aggregation of Luc in vitro. These results are in agreement; in vivo RNA interference analyses showed that a reduction of VCP level results in more aggregation of Luc in cells. Structural and functional analyses further demonstrated that the D1 domain of VCP is sufficient to mediate the aggregation-prevention activity, which does not require ATP binding, ATP hydrolysis, or a hexameric structure of VCP. Together, these results indicate that (1) VCP prevents protein aggregation in vitro and in vivo, (2) this aggregation-prevention activity is mediated mainly through the D1 domain of VCP, and (3) this activity does not require ATPase activity or a hexameric structure of VCP.     

Light-dependent phosphorylation of THRUMIN1 regulates its association with actin filaments and 14-3-3 proteins

Light-dependent chloroplast movements in leaf cells contribute to the optimization of photosynthesis. Low-light conditions induce chloroplast accumulation along periclinal cell surfaces, providing greater access to available light, whereas high light induces movement of chloroplasts to anticlinal cell surfaces, providing photodamage protection and allowing more light to reach underlying cell layers. The THRUMIN1 protein is required for normal chloroplast movements in Arabidopsis (Arabidopsis thaliana) and has been shown to localize at the plasma membrane and to undergo rapid light-dependent interactions with actin filaments through the N-terminal intrinsically disordered region (IDR). A predicted WASP-Homology 2 domain was found in the IDR but mutations in this domain did not disrupt localization of THRUMIN1:YFP to actin filaments. A series of other protein truncations and site-directed mutations of known and putative phosphorylation sites indicated that a phosphomimetic mutation (serine to aspartic acid) at position 170 disrupted localization of THRUMIN1 to actin filaments. However, the phosphomimetic mutant rescued the thrumin1-2 mutant phenotype for chloroplast movement and raises questions about the role of THRUMIN1’s interaction with actin. Mutation of serine 146 to aspartic acid also resulted in cytoplasmic localization of THRUMIN1:YFP in Nicotiana benthamiana. Mutations to a group of putative zinc-binding cysteine clusters implicate the C-terminus of THRUMIN1 in chloroplast movement. Phosphorylation-dependent association of THRUMIN1 with 14-3-3 KAPPA and OMEGA were also identified. Together, these studies provide insights into the mechanistic role of THRUMIN1 in light-dependent chloroplast movements.

Site-directed mutagenesis of THRUMIN1 revealed sites critical to its blue-light-dependent localization to actin filaments, to 14-3-3 proteins, and for its regulation of chloroplast movement.     

CD45 regulates tyrosine phosphorylation of CD22 and its association with the protein tyrosine phosphatase SHP-1

Cross-linking of CD45 induced capping and physical sequestration from CD22 leading to an increase in tyrosine phosphorylation of CD22 and SHP-1 recruitment. Additionally, CD22 isolated from a CD45-deficient B cell line exhibited increased basal/inducible tyrosine phosphorylation and enhanced recruitment of SHP-1 compared with CD22 isolated from CD45-positive parental cells. Subsequent experiments were performed to determine whether enhanced SHP-1 recruitment to CD22 is responsible for attenuation of receptor-mediated Ca2+ responses in CD45-deficient cells. Catalytically inactive SHP-1 expressed in CD45-deficient cells interacted with CD22 and decreased phosphatase activity in CD22 immunoprecipitates to levels that were comparable to those in CD45-positive cells. Expression of catalytically inactive SHP-1 restored intracellular mobilization of Ca2+ in response to MHC class II cross-linking, but did not affect B cell Ag receptor- or class II-mediated Ca2+ influx from the extracellular space. These results indicate that CD45 regulates tyrosine phosphorylation of CD22 and binding of SHP-1. The data further indicate that enhanced recruitment and activation of SHP-1 in CD45-deficient cells affect intracellular mobilization of Ca2+, but are not responsible for abrogation of receptor-mediated Ca2+ influx from the extracellular space.     

Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation

Before fertilization can occur, mammalian sperm must undergo capacitation, a process that requires a cyclic AMP-dependent increase in tyrosine phosphorylation. To identify proteins phosphorylated during capacitation, two-dimensional gel analysis coupled to anti-phosphotyrosine immunoblots and tandem mass spectrometry (MS/MS) was performed. Among the protein targets, valosin-containing protein (VCP), a homolog of the SNARE-interacting protein NSF, and two members of the A kinase-anchoring protein (AKAP) family were found to be tyrosine phosphorylated during capacitation. In addition, immobilized metal affinity chromatography was used to investigate phosphorylation sites in whole protein digests from capacitated human sperm. To increase this chromatographic selectivity for phosphopeptides, acidic residues in peptide digests were converted to their respective methyl esters before affinity chromatography. More than 60 phosphorylated sequences were then mapped by MS/MS, including precise sites of tyrosine and serine phosphorylation of the sperm tail proteins AKAP-3 and AKAP-4. Moreover, differential isotopic labeling was developed to quantify phosphorylation changes occurring during capacitation. The phosphopeptide enrichment and quantification methodology coupled to MS/MS, described here for the first time, can be employed to map and compare phosphorylation sites involved in multiple cellular processes. Although we were unable to determine the exact site of phosphorylation of VCP, we did confirm, using a cross-immunoprecipitation approach, that this protein is tyrosine phosphorylated during capacitation. Immunolocalization of VCP showed fluorescent staining in the neck of noncapacitated sperm. However, after capacitation, staining in the neck decreased, and most of the sperm showed fluorescent staining in the anterior head.     

Mad2 phosphorylation regulates its association with Mad1 and the APC/C

Improper attachment of the mitotic spindle to the kinetochores of paired sister chromatids in mitosis is monitored by a checkpoint that leads to an arrest in early metaphase. This arrest requires the inhibitory association of Mad2 with the anaphase promoting complex/cyclosome (APC/C). It is not known how the association of Mad2 with the kinetochore and the APC/C is regulated in mitosis. Here, we demonstrate that human Mad2 is modified through phosphorylation on multiple serine residues in vivo in a cell cycle dependent manner and that only unphosphorylated Mad2 interacts with Mad1 or the APC/C in vivo. A Mad2 mutant containing serine to aspartic acid mutations mimicking the C-terminal phosphorylation events fails to interact with Mad1 or the APC/C and acts as a dominant-negative antagonist of wild-type Mad2. These data suggest that the phosphorylation state of Mad2 regulates its checkpoint activity by modulating its association with Mad1 and the APC/C.     

Complete structure of p97/valosin-containing protein reveals communication between nucleotide domains

The ATPase p97/VCP affects multiple events within the cell. These events include the alteration of both nuclear and mitotic Golgi membranes, the dislocation of ubiquitylated proteins from the endoplasmic reticulum and regulation of the NF-kappa b pathway. Here we present the crystal structure of full-length Mus musculus p97/VCP in complex with a mixture of ADP and ADP-AlF(x) at a resolution of 4.7 A. This is the first complete hexameric structure of a protein containing tandem AAA (ATPases associated with a variety of cellular activities) domains. Comparison of the crystal structure and cryo-electron microscopy (EM) reconstructions reveals large conformational changes in the helical subdomains during the hydrolysis cycle. Structural and functional data imply a communication mechanism between the AAA domains. A Zn(2+) occludes the central pore of the hexamer, suggesting that substrate does not thread through the pore of the molecule.     

Tuberin phosphorylation regulates its interaction with hamartin. Two proteins involved in tuberous sclerosis

Hamartin and tuberin are products of the tumor suppressor genes, TSC1 and TSC2, respectively. When mutated, a characteristic spectrum of tumor-like growths develop resulting in the syndrome of tuberous sclerosis complex. The phenotypes associated with TSC1 and TSC2 mutations are largely indistinguishable suggesting a common biochemical pathway. Indeed, hamartin and tuberin have been shown to interact stably in vitro and in vivo. Factors that regulate their interaction are likely critical to the understanding of disease pathogenesis. In this study, we showed that tuberin is phosphorylated at serine and tyrosine residues in response to serum and other factors, and it undergoes serial phosphorylation that can be detected by differences in electrophoretic mobilities. A disease-related TSC2 mutation (Y1571H) nearly abolished tuberin phosphorylation when stimulated with pervanadate. Expression of this mutant tuberin caused a marked reduction in TSC1-TSC2 interaction compared with wild-type protein and significantly curtailed the growth inhibitory effects of tuberin when overexpressed in COS1 cells, consistent with a loss of function mutation. Examination of a second pathologic mutation, P1675L, revealed a similar relationship between limited phosphorylation and reduced interaction with hamartin. Our data show for the first time that 1) tuberin is phosphorylated at tyrosine and serine residues, 2) TSC1-TSC2 interaction is regulated by tuberin phosphorylation, and 3) defective phosphorylation of tuberin is associated with loss of its tumor suppressor activity. These findings suggest that phosphorylation may be a key regulatory mechanism controlling TSC1-TSC2 function.     

CaMKII-dependent phosphorylation regulates SAP97/NR2A interaction

Synapse-associated protein 97 (SAP97), a member of membrane-associated guanylate kinase protein family, has been implicated in the processes of targeting ionotropic glutamate receptors at postsynaptic sites. Here we show that SAP97 is enriched at the postsynaptic density where it co-localizes with both ionotropic glutamate receptors and downstream signaling proteins such as Ca2+/calmodulin-dependent protein kinase II (CaMKII). SAP97 and alphaCaMKII display a high co-localization pattern in hippocampal neurons as well as in transfected COS-7 cells. Metabolic labeling of hippocampal cultures reveals that N-methyl-D-aspartic acid (NMDA) receptor activation induces CaMKII-dependent phosphorylation of SAP97; co-incubation with the CaMKII-specific inhibitor KN-93 reduces SAP97 phosphorylation to basal levels. Our results show that SAP97 directly interacts with the NR2A subunit of NMDA receptor both in an in vitro "pull-out" assay and in co-immunoprecipitation experiments from homogenates and synaptosomes purified from hippocampal rat tissue. Interestingly, in the postsynaptic density fraction, SAP97 fails to co-precipitate with NR2A. We show here that SAP97 is directly associated with NR2A through its PDZ1 domain, and CaMKII-dependent phosphorylation of SAP97-Ser-232 disrupts NR2A interaction both in an in vitro pull-out assay and in transfected COS-7 cells. Moreover, expression of SAP97(S232D) mutant has effects similar to those observed upon constitutively activating CaMKII. Our findings suggest that SAP97/NR2A interaction is regulated by CaMKII-dependent phosphorylation and provide a novel mechanism for the regulation of synaptic targeting of NMDA receptor subunits.     

A novel function of VCP (valosin-containing protein; p97) in the control of N-glycosylation of proteins in the endoplasmic reticulum

alpha-Chain of T-cell receptor (TCR) is a typical ERAD (ER-associated degradation) substrate degraded in the absence of other TCR subunits. Depletion of derlin 1 fails to induce accumulation of alphaTCR despite inducing accumulation of alpha1-antitrypsin, another ERAD substrate. Furthermore, while depletion of VCP does not affect levels of alpha1-antitrypsin, it induces an increase in levels of alphaTCR. RNAi of VCP induces preferential accumulation of alphaTCR with less mannose residues, suggesting its retention within the ER. Mass spectrometric analysis of cellular N-linked glycans revealed that depletion of VCP decreases the level of high-mannose glycoproteins, increases the levels of truncated low-mannose glycoproteins and induces changes in the abundance of complex glycans assembled in post-ER compartments. Since proteasome inhibition was unable to mimic those changes, they cannot be regarded as a simple consequence of inhibited ERAD but represent a complex effect of VCP on the function of the ER.     

2-Anilino-4-aryl-1,3-thiazole inhibitors of valosin-containing protein (VCP or p97)

Valosin-containing protein (VCP; also known as p97) is a member of the AAA ATPase family with a central role in the ubiquitin-degradation of misfolded proteins. VCP also exhibits antiapoptotic function and metastasis via activation of nuclear factor kappa-B signaling pathway. We have discovered that 2-anilino-4-aryl-1,3-thiazoles are potent drug-like inhibitors of this enzyme. The identified compounds show low nanomolar VCP potency, demonstrate SAR trends, and show activity in a mechanism based cellular assay. This series of compounds represents the first steps towards a novel, small molecule VCP inhibitor as a cancer therapeutic.     

AAA ATPase p97/valosin-containing protein interacts with gp78, a ubiquitin ligase for endoplasmic reticulum-associated degradation

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control mechanism that eliminates unwanted proteins from the endoplasmic reticulum (ER) through a ubiquitin-dependent proteasomal degradation pathway. gp78 is a previously described ER membrane-anchored ubiquitin ligase (E3) involved in ubiquitination of ER proteins. AAA ATPase (ATPase associated with various cellular activities) p97/valosin-containing protein (VCP) subsequently dislodges the ubiquitinated proteins from the ER and chaperones them to the cytosol, where they undergo proteasomal degradation. We now report that gp78 physically interacts with p97/VCP and enhances p97/VCP-polyubiquitin association. The enhanced association correlates with decreases in ER stress-induced accumulation of polyubiquitinated proteins. This effect is abolished when the p97/VCP-interacting domain of gp78 is removed. Further, using ERAD substrate CD3delta, gp78 consistently enhances p97/VCP-CD3delta binding and facilitates CD3delta degradation. Moreover, inhibition of endogenous gp78 expression by RNA interference markedly increases the levels of total polyubiquitinated proteins, including CD3delta, and abrogates VCP-CD3delta interactions. The gp78 mutant with deletion of its p97/VCP-interacting domain fails to increase CD3delta degradation and leads to accumulation of polyubiquitinated CD3delta, suggesting a failure in delivering ubiquitinated CD3delta for degradation. These data suggest that gp78-p97/VCP interaction may represent one way of coupling ubiquitination with retrotranslocation and degradation of ERAD substrates.     

RNA-binding protein RBM14 regulates dissociation and association of non-homologous end joining proteins

Defects in the DNA damage response (DDR) are associated with multiple diseases, including cancers and neurodegenerative disorders. Emerging evidence indicates involvement of RNA-binding proteins (RBPs) in DDR. However, functions of RBPs in the DDR pathway remain elusive. We have shown previously that the RNA-binding protein RBM14 is required for non-homologous end joining (NHEJ). Here we show that RBM14 is required for efficient recruitment of XRCC4 and XLF to chromatin and the release of KU proteins from chromatin upon DNA damage. Failure of this process leads to accumulation of double-strand breaks (DSBs) in cells. Thus RBM14 plays crucial role in regulation of NHEJ upon DNA damage.     

Plk phosphorylation regulates the microtubule-stabilizing protein TCTP

The mitotic polo-like kinases have been implicated in the formation and function of bipolar spindles on the basis of their respective localizations and mutant phenotypes. To date, this putative regulation has been limited to a kinesin-like motor protein, a centrosomal structural protein, and two microtubule-associated proteins (MAPs). In this study, another spindle-regulating protein, the mammalian non-MAP microtubule-binding and -stabilizing protein, the translationally controlled tumor protein (TCTP), was identified as a putative Plk-interacting clone by a two-hybrid screen. Plk phosphorylates TCTP on two serine residues in vitro and cofractionates with the majority of kinase activity toward TCTP in mitotic cell lysates. In addition, these sites were demonstrated to be phosphorylated in vivo. Overexpression of a Plk phosphorylation site-deficient mutant of TCTP induced a dramatic increase in the number of multinucleate cells, rounded cells with condensed ball-like nuclei, and cells undergoing cell death, similar to both the reported anti-Plk antibody microinjection and the low-concentration taxol treatment phenotypes. These results suggest that phosphorylation decreases the microtubule-stabilizing activity of TCTP and promotes the increase in microtubule dynamics that occurs after metaphase.