Tyrosine Phosphorylation of Growth Factor Receptor-bound Protein-7 by Focal Adhesion Kinase in the Regulation of Cell Migration, Proliferation, and Tumorigenesis

We have previously reported that growth factor receptor-bound protein-7 (Grb7), an Src-homology 2 (SH2)-containing adaptor protein, enables interaction with focal adhesion kinase (FAK) to regulate cell migration in response to integrin activation. To further elucidate the signaling events mediated by FAK·Grb7 complexes in promoting cell migration and other cellular functions, we firstly examined the phos pho ryl a ted tyrosine site(s) of Grb7 by FAK using an in vivo mutagenesis. We found that FAK was capable of phos pho rylating at least 2 of 12 tyrosine residues within Grb7, Tyr-188 and Tyr-338. Moreover, mutations converting the identified Tyr to Phe inhibited integrin-dependent cell migration as well as impaired cell proliferation but not survival compared with the wild-type control. Interestingly, the above inhibitory effects caused by the tyrosine phos pho ryl a tion-deficient mutants are probably attributed to their down-regulation of phospho-Tyr-397 of FAK, thereby implying a mechanism by competing with wild-type Grb7 for binding to FAK. Consequently, these tyrosine phos pho ryl a tion-deficient mutants evidently altered the phospho-Tyr-118 of paxillin and phos pho ryl a tion of ERK1/2 but less on phospho-Ser-473 of AKT, implying their involvement in the FAK·Grb7-mediated cellular functions. Additionally, we also illustrated that the formation of FAK·Grb7 complexes and Grb7 phos pho ryl a tion by FAK in an integrin-dependent manner were essential for cell migration, proliferation and anchorage-independent growth in A431 epidermal carcinoma cells, indicating the importance of FAK·Grb7 complexes in tumorigenesis. Our data provide a better understanding on the signal transduction event for FAK·Grb7-mediated cellular functions as well as to shed light on a potential therapeutic in cancers.Growth factor receptor bound protein-7 (Grb7)² is initially identified as a SH2 domain-containing adaptor protein bound to the activated EGF receptor (1). Grb7 is composed of an N-terminal proline-rich region, following a putative RA (Ras-associating) domain and a central PH (pleckstrin homology) domain and a BPS motif (between PH and SH2 domains), and a C-terminal SH2 domain (2–6). Despite the lack of enzymatic activity, the presence of multiple protein-protein interaction domains allows Grb7 family adaptor proteins to participate in versatile signal transduction pathways and, therefore, to regulate many cellular functions (4–6). A number of signaling molecules has been reported to interact with these featured domains, although most of the identified Grb7 binding partners are mediated through its SH2 domain. For example, the SH2 domain of Grb7 has been demonstrated to be capable of binding to the phospho-tyrosine sites of EGF receptor (1), ErbB2 (7), ErbB3 and ErbB4 (8), Ret (9), platelet-derived growth factor receptor (10), insulin receptor (11), SHPTP2 (12), Tek/Tie2 (13), caveolin (14), c-Kit (15), EphB1 (16), G6f immunoreceptor protein (17), Rnd1 (18), Shc (7), FAK (19), and so on. The proceeding α-helix of the PH domain of Grb7 is the calmodulin-binding domain responsible for recruiting Grb7 to plasma membrane in a Ca²⁺-dependent manner (20), and the association between the PH domain of Grb7 and phosphoinositides is required for the phosphorylation by FAK (21). Two additional proteins, NIK (nuclear factor κB-inducing kinase) and FHL2 (four and half lim domains isoform 2), in association with the GM region (Grb and Mig homology region) of Grb7 are also reported, although the physiological functions for these interactions remain unknown (22, 23). Recently, other novel roles in translational controls and stress responses through the N terminus of Grb7 are implicated for the findings of Grb7 interacting with the 5′-untranslated region of capped targeted KOR (kappa opioid receptor) mRNA and the Hu antigen R of stress granules in an FAK-mediated phosphorylation manner (24, 25).Unlike its member proteins Grb10 and Grb14, the role of Grb7 in cell migration is unambiguous and well documented. This is supported by a series of studies. Firstly, Grb7 family members share a significantly conserved molecular architecture with the Caenorhabditis elegans Mig-10 protein, which is involved in neuronal cell migration during embryonic development (4, 5, 26), suggesting that Grb7 may play a role in cell migration. Moreover, Grb7 is often co-amplified with Her2/ErbB2 in certain human cancers and tumor cell lines (7, 27, 28), and its overexpression resulted in invasive and metastatic consequences of various cancers and tumor cells (23, 29–33). On the contrary, knocking down Grb7 by RNA interference conferred to an inhibitory outcome of the breast cancer motility (34). Furthermore, interaction of Grb7 with autophosphorylated FAK at Tyr-397 could promote integrin-mediated cell migration in NIH 3T3 and CHO cells, whereas overexpression of its SH2 domain, an dominant negative mutant of Grb7, inhibited cell migration (19, 35). Recruitment and phosphorylation of Grb7 by EphB1 receptors enhanced cell migration in an ephrin-dependent manner (16). Recently, G7–18NATE, a selective Grb7-SH2 domain affinity cyclic peptide, was demonstrated to efficiently block cell migration of tumor cells (32, 36). In addition to cell migration, Grb7 has been shown to play a role in a variety of physiological and pathological events, for instance, kidney development (37), tumorigenesis (7, 14, 38–41), angiogenic activity (20), proliferation (34, 42, 43), anti-apoptosis (44), gene expression regulation (24), Silver-Russell syndrome (45), rheumatoid arthritis (46), atopic dermatitis (47), and T-cell activation (17, 48). Nevertheless, it remains largely unknown regarding the downstream signaling events of Grb7-mediated various functions. In particular, given the role of Grb7 as an adaptor molecule and its SH2 domain mainly interacting with upstream regulators, it will be interesting to identify potential downstream effectors through interacting with the functional GM region or N-terminal proline-rich region.In this report, we identified two tyrosine phosphorylated sites of Grb7 by FAK and deciphered the signaling targets downstream through these phosphorylated tyrosine sites to regulate various cellular functions such as cell migration, proliferation, and survival. In addition, our study sheds light on tyrosine phosphorylation of Grb7 by FAK involved in tumorigenesis.     

PTIP Regulates 53BP1 and SMC1 at the DNA Damage Sites

Although PTIP is implicated in the DNA damage response, through interactions with 53BP1, the function of PTIP in the DNA damage response remain elusive. Here, we show that RNF8 controls DNA damage-induced nuclear foci formation of PTIP, which in turn regulates 53BP1 localization to the DNA damage sites. In addition, SMC1, a substrate of ATM, could not be phosphorylated at the DNA damage sites in the absence of PTIP. The PTIP-dependent pathway is important for DNA double strand breaks repair and DNA damage-induced intra-S phase checkpoint activation. Taken together, these results suggest that the role of PTIP in the DNA damage response is downstream of RNF8 and upstream of 53BP1. Thus, PTIP regulates 53BP1-dependent signaling pathway following DNA damage.The DNA damage response pathways are signal transduction pathways with DNA damage sensors, mediators, and effectors, which are essential for maintaining genomic stability (1–3). Following DNA double strand breaks, histone H2AX at the DNA damage sites is rapidly phosphorylated by ATM/ATR/DNAPK (4–10), a family homologous to phosphoinositide 3-kinases (11, 12). Subsequently, phospho-H2AX (γH2AX) provides the platform for accumulation of a larger group of DNA damage response factors, such as MDC1, BRCA1, 53BP1, and the MRE11·RAD50·NBS1 complex (13, 14), at the DNA damage sites. Translocalization of these proteins to the DNA double strand breaks (DSBs)³ facilitates DNA damage checkpoint activation and enhances the efficiency of DNA damage repair (14, 15).Recently, PTIP (Pax2 transactivation domain-interacting protein, or Paxip) has been identified as a DNA damage response protein and is required for cell survival when exposed to ionizing radiation (IR) (1, 16–18). PTIP is a 1069-amino acid nuclear protein and has been originally identified in a yeast two-hybrid screening as a partner of Pax2 (19). Genetic deletion of the PTIP gene in mice leads to early embryonic lethality at embryonic day 8.5, suggesting that PTIP is essential for early embryonic development (20). Structurally, PTIP contains six tandem BRCT (BRCA1 carboxyl-terminal) domains (16–18, 21). The BRCT domain is a phospho-group binding domain that mediates protein-protein interactions (17, 22, 23). Interestingly, the BRCT domain has been found in a large number of proteins involved in the cellular response to DNA damages, such as BRCA1, MDC1, and 53BP1 (7, 24–29). Like other BRCT domain-containing proteins, upon exposure to IR, PTIP forms nuclear foci at the DSBs, which is dependent on its BRCT domains (16–18). By protein affinity purification, PTIP has been found in two large complexes. One includes the histone H3K4 methyltransferase ALR and its associated cofactors, the other contains DNA damage response proteins, including 53BP1 and SMC1 (30, 31). Further experiments have revealed that DNA damage enhances the interaction between PTIP and 53BP1 (18, 31).To elucidate the DNA damage response pathways, we have examined the upstream and downstream partners of PTIP. Here, we report that PTIP is downstream of RNF8 and upstream of 53BP1 in response to DNA damage. Moreover, PTIP and 53BP1 are required for the phospho-ATM association with the chromatin, which phosphorylates SMC1 at the DSBs. This PTIP-dependent pathway is involved in DSBs repair.     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

Biochemical Characterization and Function of Complexes Formed by Hyaluronan and the Heavy Chains of Inter-��-inhibitor (HC��HA) Purified from Extracts of Human Amniotic Membrane

Clinically, amniotic membrane (AM) suppresses inflammation, scarring, and angiogenesis. AM contains abundant hyaluronan (HA) but its function in exerting these therapeutic actions remains unclear. Herein, AM was extracted sequentially with buffers A, B, and C, or separately by phosphate-buffered saline (PBS) alone. Agarose gel electrophoresis showed that high molecular weight (HMW) HA (an average of ∼3000 kDa) was predominantly extracted in isotonic Extract A (70.1 ± 6.0%) and PBS (37.7 ± 3.2%). Western blot analysis of these extracts with hyaluronidase digestion or NaOH treatment revealed that HMW HA was covalently linked with the heavy chains (HCs) of inter-α-inhibitor (IαI) via a NaOH-sensitive bond, likely transferred by the tumor necrosis factor-α stimulated gene-6 protein (TSG-6). This HC·HA complex (nHC·HA) could be purified from Extract PBS by two rounds of CsCl/guanidine HCl ultracentrifugation as well as in vitro reconstituted (rcHC·HA) by mixing HMW HA, serum IαI, and recombinant TSG-6. Consistent with previous reports, Extract PBS suppressed transforming growth factor-β1 promoter activation in corneal fibroblasts and induced mac ro phage apo pto sis. However, these effects were abolished by hyaluronidase digestion or heat treatment. More importantly, the effects were retained in the nHC·HA or rcHC·HA. These data collectively suggest that the HC·HA complex is the active component in AM responsible in part for clinically observed anti-inflammatory and anti-scarring actions.Hyaluronan (HA)⁴ is widely distributed in extracellular matrices, tissues, body fluids, and even in intracellular compartments (reviewed in Refs. 1 and 2). The molecular weight of HA ranges from 200 to 10,000 kDa depending on the source (3), but can also exist as smaller fragments and oligosaccharides under certain physiological or pathological conditions (1). Investigations over the last 15 years have suggested that low M_r HA can induce the gene expression of proinflammatory mediators and proangiogenesis, whereas high molecular weight (HMW) HA inhibits these processes (4–7).Several proteins have been shown to bind to HA (8) such as aggrecan (9), cartilage link protein (10), versican (11), CD44 (12, 13), inter-α-inhibitor (IαI) (14, 15), and tumor necrosis factor-α stimulated gene-6 protein (TSG-6) (16, 17). IαI consists of two heavy chains (HCs) (HC1 and HC2), both of which are linked through ester bonds to a chondroitin sulfate chain that is attached to the light chain, i.e. bikunin. Among all HA-binding proteins, only the HCs of IαI have been clearly demonstrated to be covalently coupled to HA (14, 18). However, TSG-6 has also been reported to form stable, possibly covalent, complexes with HA, either alone (19, 20) or when associated with HC (21).The formation of covalent bonds between HCs and HA is mediated by TSG-6 (22–24) where its expression is often induced by inflammatory mediators such as tumor necrosis factor-α and interleukin-1 (25, 26). TSG-6 is also expressed in inflammatory-like processes, such as ovulation (21, 27, 28) and cervical ripening (29). TSG-6 interacts with both HA (17) and IαI (21, 24, 30–33), and is essential for covalently transferring HCs on to HA (22–24). The TSG-6-mediated formation of the HC·HA complex has been demonstrated to play a crucial role in female fertility in mice. The HC·HA complex is an integral part of an expanded extracellular “cumulus” matrix around the oocyte, which plays a critical role in successful ovulation and fertilization in vivo (22, 34). HC·HA complexes have also been found at sites of inflammation (35–38) where its pro- or anti-inflammatory role remain arguable (39, 40).Immunostaining reveals abundant HA in the avascular stromal matrix of the AM (41, 42).⁵ In ophthalmology, cryopreserved AM has been widely used as a surgical graft for ocular surface reconstruction and exerts clinically observable actions to promote epithelial wound healing and to suppress inflammation, scarring, and angiogenesis (for reviews see Refs. 43–45). However, it is not clear whether HA in AM forms HC·HA complex, and if so whether such an HC·HA complex exerts any of the above therapeutic actions. To address these questions, we extracted AM with buffers of increasing salt concentration. Because HMW HA was found to form the HC·HA complex and was mainly extractable by isotonic solutions, we further purified it from the isotonic AM extract and reconstituted it in vitro from three defined components, i.e. HMW HA, serum IαI, and recombinant TSG-6. Our results showed that the HC·HA complex is an active component in AM responsible for the suppression of TGF-β1 promoter activity, linkable to the scarring process noted before by AM (46–48) and by the AM soluble extract (49), as well as for the promotion of macrophage death, linkable to the inflammatory process noted by AM (50) and the AM soluble extract (51).     

Mcl-1 Degradation during Hepatocyte Lipoapoptosis

The mechanisms of free fatty acid-induced lipoapoptosis are incompletely understood. Here we demonstrate that Mcl-1, an anti-apoptotic member of the Bcl-2 family, was rapidly degraded in hepatocytes in response to palmitate and stearate by a proteasome-dependent pathway. Overexpression of a ubiquitin-resistant Mcl-1 mutant in Huh-7 cells attenuated palmitate-mediated Mcl-1 loss and lipoapoptosis; conversely, short hairpin RNA-targeted knockdown of Mcl-1 sensitized these cells to lipoapoptosis. Palmitate-induced Mcl-1 degradation was attenuated by the novel protein kinase C (PKC) inhibitor rottlerin. Of the two human novel PKC isozymes, PKCδ and PKCθ, only activation of PKCθ was observed by phospho-immunoblot analysis. As compared with Jurkat cells, a smaller PKCθ polypeptide and mRNA were expressed in hepatocytes consistent with an alternative splice variant. Short hairpin RNA-mediated knockdown of PKCθ reduced Mcl-1 degradation and lipoapoptosis. Likewise, genetic deletion of Pkcθ also attenuated Mcl-1 degradation and cytotoxicity by palmitate in primary hepatocytes. During treatment with palmitate, rottlerin inhibited phosphorylation of Mcl-1 at Ser¹⁵⁹, a phosphorylation site previously implicated in Mcl-1 turnover. Consistent with these results, an Mcl-1 S159A mutant was resistant to degradation and improved cell survival during palmitate treatment. Collectively, these results implicate PKCθ-dependent destabilization of Mcl-1 as a mechanism contributing to hepatocyte lipoapoptosis.Current evidence suggests that hepatic steatosis is present in up to 30% of the American population (1). A subset of these individuals develop severe hepatic lipotoxicity, a syndrome referred to as NASH² (2), which can progress to cirrhosis and its chronic sequela (3, 4). A major risk factor for hepatic lipotoxicity is insulin resistance (5–7), resulting in excessive lipolysis within peripheral adipose tissue with release of high levels of free fatty acids (FFA) to the circulation. Circulating FFA are taken up by the liver via fatty acid transporter 5 and CD36 (8–10), and the bulk of hepatic neutral fat is derived from re-esterification of circulating FFA (8). Current concepts indicate that FFA, and not their esterified product (triglyceride), mediate hepatic lipotoxicity (11, 12). Elevated serum FFA correlate with liver disease severity (13–15), and therapies that enhance insulin sensitivity ameliorate hepatic lipotoxicity, in part, by decreasing plasma FFA (16). Hepatic FFA also accumulate in experimental steatohepatitis, further supporting a role for these nutrients in hepatic lipotoxicity (17). Saturated FFA are more strongly implicated in hepatic lipotoxicity than unsaturated FFA (18, 19). Saturated FFA induce hepatocyte apoptosis (20, 21), a cardinal feature of nonalcoholic fatty liver disease (22), and serum biomarkers of apoptosis are useful for identifying hepatic lipotoxicity (23). Thus, FFA-mediated lipotoxicity occurs, in part, by apoptosis.Apoptosis is regulated by members of the Bcl-2 protein family (24). These proteins can be categorized into three subsets as follows: the guardians or anti-apoptotic members of this family, which include Bcl-2, A1, Mcl-1, Bcl-x_L, and Bcl-w; the multidomain executioners or proapoptotic members of this family, which include Bax and Bak; and the messengers or biosensors of cell death, which share only the third Bcl-2 homology domain and are referred to as BH3-only proteins. This last group of proteins includes Bid, Bim, Bmf, Puma, Noxa, Hrk, Bad, and Bik. We have previously reported that cytotoxic FFA induce Bim expression by a FoxO3a-dependent mechanism that contributes, in part, to lipoapoptosis by activating Bax (20, 21). However, Bax activation can be held in check by anti-apoptotic members of the Bcl-2 family suggesting their function may also be dysregulated during FFA-mediated cytotoxicity.Bcl-2 is not expressed in hepatocytes at the protein level (25), whereas Bcl-w and Bfl-1/A1 knock-out mice have no liver phenotype (26–28). However, both potent anti-apoptotic proteins Bcl-x_L and Mcl-1 are expressed by hepatocytes and exhibit a liver phenotype in knock-out mice (29, 30), whereas up-regulation of Mcl-1 renders hepatocytes resistant to apoptosis (31–33). It has also been posited that cellular elimination of Mcl-1 is a critical step in certain proapoptotic cascades (34, 35). Mcl-1 is unique among Bcl-2 proteins in that it has a short half-life, 30–120 min in most cell types, due to the presence of two sequences rich in proline, glutamic acid, serine, and threonine, which target the protein for rapid degradation by the proteasome (36). Proteasomal degradation of Mcl-1 is promoted by ubiquitination, which in turn is regulated by various kinase cascades (36). Despite its potential importance, a role for Mcl-1 in regulating hepatocyte FFA-mediated lipoapoptosis remains unexplored.Given that FFA induce insulin resistance (37), the kinases potentially regulating lipoapoptosis are likely those also identified in insulin resistance syndromes, especially the novel PKC isoforms PKCδ and PKCθ (38). The novel PKC isoforms are activated by diacylglycerol, which rises in the presence of FFA (39–41), and diacylglycerol levels are significantly increased in NASH (42). A role for PKCδ in apoptosis has not been described. PKCθ has recently been shown to be activated by endoplasmic reticulum stress in liver cells (43) and lipids in vivo (44, 45). Furthermore, PKCθ has also been implicated in apoptosis of Jurkat cells, neuroblastoma cells, and myeloid leukemia cells (46, 47). However, neither its role in mediating lipoapoptosis nor modulating levels/activity of Bcl-2 proteins has been examined.This study addresses the role of Mcl-1 and PKCθ in FFA-induced lipoapoptosis. We identify a pathway that involves PKCθ-dependent proteasomal degradation of Mcl-1. Using inhibitors of various steps along this pathway, along with Mcl-1 mutants that are resistant to proteasomal degradation or Ser¹⁵⁹ phosphorylation, our studies implicate Mcl-1 degradation via a PKCθ-dependent process as a critical step in lipoapoptosis.     

A Pipeline for Determining Protein–Protein Interactions and Proximities in the Cellular Milieu

It remains extraordinarily challenging to elucidate endogenous protein-protein interactions and proximities within the cellular milieu. The dynamic nature and the large range of affinities of these interactions augment the difficulty of this undertaking. Among the most useful tools for extracting such information are those based on affinity capture of target bait proteins in combination with mass spectrometric readout of the co-isolated species. Although highly enabling, the utility of affinity-based methods is generally limited by difficulties in distinguishing specific from nonspecific interactors, preserving and isolating all unique interactions including those that are weak, transient, or rapidly exchanging, and differentiating proximal interactions from those that are more distal. Here, we have devised and optimized a set of methods to address these challenges. The resulting pipeline involves flash-freezing cells in liquid nitrogen to preserve the cellular environment at the moment of freezing; cryomilling to fracture the frozen cells into intact micron chunks to allow for rapid access of a chemical reagent and to stabilize the intact endogenous subcellular assemblies and interactors upon thawing; and utilizing the high reactivity of glutaraldehyde to achieve sufficiently rapid stabilization at low temperatures to preserve native cellular interactions. In the course of this work, we determined that relatively low molar ratios of glutaraldehyde to reactive amines within the cellular milieu were sufficient to preserve even labile and transient interactions. This mild treatment enables efficient and rapid affinity capture of the protein assemblies of interest under nondenaturing conditions, followed by bottom-up MS to identify and quantify the protein constituents. For convenience, we have termed this approach Stabilized Affinity Capture Mass Spectrometry. Here, we demonstrate that Stabilized Affinity Capture Mass Spectrometry allows us to stabilize and elucidate local, distant, and transient protein interactions within complex cellular milieux, many of which are not observed in the absence of chemical stabilization.Insights into many cellular processes require detailed information about interactions between the participating proteins. However, the analysis of such interactions can be challenging because of the often-diverse physicochemical properties and the abundances of the constituent proteins, as well as the sometimes wide range of affinities and complex dynamics of the interactions. One of the key challenges has been acquiring information concerning transient, low affinity interactions in highly complex cellular milieux (3, 4).Methods that allow elucidation of such information include co-localization microscopy (5), fluorescence protein Förster resonance energy transfer (4), immunoelectron microscopy (5), yeast two-hybrid (6), and affinity capture (7, 8). Among these, affinity capture (AC)¹ has the unique potential to detect all specific in vivo interactions simultaneously, including those that interact both directly and indirectly. In recent times, the efficacy of such affinity isolation experiments has been greatly enhanced through the use of sensitive modern mass spectrometric protein identification techniques (9). Nevertheless, AC suffers from several shortcomings. These include the problem of 1) distinguishing specific from nonspecific interactors (10, 11); 2) preserving and isolating all unique interactions including those that are weak and/or transient, as well as those that exchange rapidly (10, 12, 13); and 3) differentiating proximal from more distant interactions (14).We describe here an approach to address these issues, which makes use of chemical stabilization of protein assemblies in the complex cellular milieu prior to AC. Chemical stabilization is an emerging technique for stabilizing and elucidating protein associations both in vitro (15–20) and in vivo (3, 12, 14, 21–29), with mass spectrometric (MS) readout of the AC proteins and their connectivities. Such chemical stabilization methods are indeed well-established and are often used in electron microscopy for preserving complexes and subcellular structures both in the cellular milieu (3) and in purified complexes (30, 31), wherein the most reliable, stable, and established stabilization reagents is glutaraldehyde. Recently, glutaraldehyde has been applied in the “GraFix” protocol in which purified protein complexes are subjected to centrifugation through a density gradient that also contains a gradient of glutaraldehyde (30, 31), allowing for optimal stabilization of authentic complexes and minimization of nonspecific associations and aggregation. GraFix has also been combined with mass spectrometry on purified complexes bound to EM grids to obtain a compositional analysis of the complexes (32), thereby raising the possibility that glutaraldehyde can be successfully utilized in conjunction with AC in complex cellular milieux directly.In this work, we present a robust pipeline for determining specific protein-protein interactions and proximities from cellular milieux. The first steps of the pipeline involve the well-established techniques of flash freezing the cells of interest in liquid nitrogen and cryomilling, which have been known for over a decade (33, 34) to preserve the cellular environment, as well as having shown outstanding performance when used in analysis of macromolecular interactions in yeast (35–39), bacterial (40, 41), trypanosome (42), mouse (43), and human (44–47) systems. The resulting frozen powder, composed of intact micron chunks of cells that have great surface area and outstanding solvent accessibility, is well suited for rapid low temperature chemical stabilization using glutaraldehyde. We selected glutaraldehyde for our procedure based on the fact that it is a very reactive stabilizing reagent, even at lower temperatures, and because it has already been shown to stabilize enzymes in their functional state (48–50). We employed highly efficient, rapid, single stage affinity capture (36, 51) for isolation and bottom-up MS for analysis of the macromolecular assemblies of interest (52–54). For convenience, we have termed this approach Stabilized Affinity-Capture Mass Spectrometry (SAC-MS).     

Sgt1 Dimerization Is Negatively Regulated by Protein Kinase CK2-mediated Phosphorylation at Ser361

The kinetochore, which consists of centromere DNA and structural proteins, is essential for proper chromosome segregation in eukaryotes. In budding yeast, Sgt1 and Hsp90 are required for the binding of Skp1 to Ctf13 (a component of the core kinetochore complex CBF3) and therefore for the assembly of CBF3. We have previously shown that Sgt1 dimerization is important for this kinetochore assembly mechanism. In this study, we report that protein kinase CK2 phosphorylates Ser³⁶¹ on Sgt1, and this phosphorylation inhibits Sgt1 dimerization.The kinetochore is a structural protein complex located in the centromeric region of the chromosome coupled to spindle microtubules (1, 2). The kinetochore generates a signal to arrest cells during mitosis when it is not properly attached to microtubules, thereby preventing chromosome missegregation, which can lead to aneuploidy (3, 4). The molecular structure of the kinetochore complex of the budding yeast Saccharomyces cerevisiae has been well characterized; it is composed of more than 70 proteins, many of which are conserved in mammals (2).The centromere DNA in the budding yeast is a 125-bp region that contains three conserved regions, CDEI, CDEII, and CDEIII (5, 6). CDEIII (25 bp) is essential for centromere function (7) and is bound to a key component of the centromere, the CBF3 complex. The CBF3 complex contains four proteins, Ndc10, Cep3, Ctf13 (8–15), and Skp1 (14, 15), all essential for viability. Mutations in any of the CBF3 proteins abolish the ability of CDEIII to bind to CBF3 (16, 17). All of the kinetochore proteins, except the CDEI-binding Cbf1 (18–20), localize to the kinetochores in a CBF3-dependent manner (2). Thus, CBF3 is a fundamental kinetochore complex, and its mechanism of assembly is of great interest.We have previously found that Sgt1 and Skp1 activate Ctf13; thus, they are required for assembly of the CBF3 complex (21). The molecular chaperone Hsp90 is also required to form the active Ctf13-Skp1 complex (22). Sgt1 has two highly conserved motifs that are required for protein-protein interaction: the tetratricopeptide repeat (21) and the CHORD protein and Sgt1-specific motif. We and others have found that both domains are important for the interaction of Sgt1 with Hsp90 (23–26), which is required for assembly of the core kinetochore complex. This interaction is an initial step in kinetochore activation (24, 26, 27), which is conserved between yeast and humans (28, 29).We have recently shown that Sgt1 dimerization is important for Sgt1-Skp1 binding and therefore for kinetochore assembly (30). In this study, we have found that protein kinase CK2 phosphorylates Sgt1 at Ser³⁶¹, and this phosphorylation inhibits Sgt1 dimerization. Therefore, CK2 appears to regulate kinetochore assembly negatively in budding yeast.     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Dbf4-Cdc7 Phosphorylation of Mcm2 Is Required for Cell Growth

The Dbf4-Cdc7 kinase (DDK) is required for the activation of the origins of replication, and DDK phosphorylates Mcm2 in vitro. We find that budding yeast Cdc7 alone exists in solution as a weakly active multimer. Dbf4 forms a likely heterodimer with Cdc7, and this species phosphorylates Mcm2 with substantially higher specific activity. Dbf4 alone binds tightly to Mcm2, whereas Cdc7 alone binds weakly to Mcm2, suggesting that Dbf4 recruits Cdc7 to phosphorylate Mcm2. DDK phosphorylates two serine residues of Mcm2 near the N terminus of the protein, Ser-164 and Ser-170. Expression of mcm2-S170A is lethal to yeast cells that lack endogenous MCM2 (mcm2Δ); however, this lethality is rescued in cells harboring the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Mcm2 is required for cell growth.The Cdc7 protein kinase is required throughout the yeast S phase to activate origins (1, 2). The S phase cyclin-dependent kinase also activates yeast origins of replication (3–5). It has been proposed that Dbf4 activates Cdc7 kinase in S phase, and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6). However, it is not known how Dbf4-Cdc7 (DDK)² acts during S phase to trigger the initiation of DNA replication. DDK has homologs in other eukaryotic species, and the role of Cdc7 in activation of replication origins during S phase may be conserved (7–10).The Mcm2-7 complex functions with Cdc45 and GINS to unwind DNA at a replication fork (11–15). A mutation of MCM5 (mcm5-bob1) bypasses the cellular requirements for DBF4 and CDC7 (16), suggesting a critical physiologic interaction between Dbf4-Cdc7 and Mcm proteins. DDK phosphorylates Mcm2 in vitro with proteins purified from budding yeast (17, 18) or human cells (19). Furthermore, there are mutants of MCM2 that show synthetic lethality with DBF4 mutants (6, 17), suggesting a biologically relevant interaction between DBF4 and MCM2. Nevertheless, the physiologic role of DDK phosphorylation of Mcm2 is a matter of dispute. In human cells, replacement of MCM2 DDK-phosphoacceptor residues with alanines inhibits DNA replication, suggesting that Dbf4-Cdc7 phosphorylation of Mcm2 in humans is important for DNA replication (20). In contrast, mutation of putative DDK phosphorylation sites at the N terminus of Schizosaccharomyces pombe Mcm2 results in viable cells, suggesting that phosphorylation of S. pombe Mcm2 by DDK is not critical for cell growth (10).In budding yeast, Cdc7 is present at high levels in G₁ and S phase, whereas Dbf4 levels peak in S phase (18, 21, 22). Furthermore, budding yeast DDK binds to chromatin during S phase (6), and it has been shown that Dbf4 is required for Cdc7 binding to chromatin in budding yeast (23, 24), fission yeast (25), and Xenopus (9). Human and fission yeast Cdc7 are inert on their own (7, 8), but Dbf4-Cdc7 is active in phosphorylating Mcm proteins in budding yeast (6, 26), fission yeast (7), and human (8, 10). Based on these data, it has been proposed that Dbf4 activates Cdc7 kinase in S phase and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6, 9, 18, 21–24). However, a mechanistic analysis of how Dbf4 activates Cdc7 has not yet been accomplished. For example, the multimeric state of the active Dbf4-Cdc7 complex is currently disputed. A heterodimer of fission yeast Cdc7 (Hsk1) in complex with fission yeast Dbf4 (Dfp1) can phosphorylate Mcm2 (7). However, in budding yeast, oligomers of Cdc7 exist in the cell (27), and Dbf4-Cdc7 exists as oligomers of 180 and 300 kDa (27).DDK phosphorylates the N termini of human Mcm2 (19, 20, 28), human Mcm4 (10), budding yeast Mcm4 (26), and fission yeast Mcm6 (10). Although the sequences of the Mcm N termini are poorly conserved, the DDK sites identified in each study have neighboring acidic residues. The residues of budding yeast Mcm2 that are phosphorylated by DDK have not yet been identified.In this study, we find that budding yeast Cdc7 is weakly active as a multimer in phosphorylating Mcm2. However, a low molecular weight form of Dbf4-Cdc7, likely a heterodimer, has a higher specific activity for phosphorylation of Mcm2. Dbf4 or DDK, but not Cdc7, binds tightly to Mcm2, suggesting that Dbf4 recruits Cdc7 to Mcm2. DDK phosphorylates two serine residues of Mcm2, Ser-164 and Ser-170, in an acidic region of the protein. Mutation of Ser-170 is lethal to yeast cells, but this phenotype is rescued by the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Ser-170 of Mcm2 is required for budding yeast growth.