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]     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[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]     

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.     

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.     

The Herpes Simplex Virus Type 1 Cleavage/Packaging Protein,UL32, Is Involved in Efficient Localization of Capsids to Replication Compartments

Six genes, including UL32, have been implicated in the cleavage and packaging of herpesvirus DNA into preassembled capsids. We have isolated a UL32 insertion mutant which is capable of near-wild-type levels of viral DNA synthesis; however, the mutant virus is unable to cleave and package viral DNA, consistent with the phenotype of a previously isolated temperature-sensitive herpes simplex virus type 1 mutant, tsN20 (P. A. Schaffer, G. M. Aron, N. Biswal, and M. Benyesh-Melnick, Virology 52:57–71, 1973). A polyclonal antibody which recognizes UL32 was previously used by Chang et al. (Y. E. Chang, A. P. Poon, and B. Roizman, J. Virol. 70:3938–3946, 1996) to demonstrate that UL32 accumulates predominantly in the cytoplasm of infected cells. In this report, a functional epitope-tagged version of UL32 showed that while UL32 is predominantly cytoplasmic, some nuclear staining which colocalizes with the major DNA binding protein (ICP8, UL29) in replication compartments can be detected. We have also used a monoclonal antibody (5C) specific for the hexon form of major capsid protein VP5 to study the distribution of capsids during infection. In cells infected with wild-type KOS (6 and 8 h postinfection), 5C staining patterns indicate that capsids are present in nuclei within replication compartments. These results suggest that cleavage and packaging occur in replication compartments at least at 6 and 8 h postinfection. Cells infected with the UL32 mutant exhibit a hexon staining pattern which is more diffusely distributed throughout the nucleus and which is not restricted to replication compartments. We propose that UL32 may play a role in “bringing” preassembled capsids to the sites of DNA packaging and that the failure to localize to replication compartments may explain the cleavage/packaging defect exhibited by this mutant. These results suggest that the UL32 protein is required at a step distinct from those at which other cleavage and packaging proteins are required and may be involved in the correct localization of capsids within infected cells.During infection of cells with herpes simplex virus type 1 (HSV-1), the large concatemeric products of DNA replication are cleaved to unit length and packaged into preassembled capsids. Capsids are icosahedral structures composed of 150 hexons and 12 pentons. Three types of capsids (A, B, and C) can be isolated from infected cells by velocity centrifugation (20). C capsids contain the viral DNA genome; B capsids contain the scaffolding protein; and A capsids contain neither DNA nor the scaffolding protein. Pulse chase experiments with another alphaherpesvirus, equine herpesvirus 1, indicate that at least some B capsids can package DNA and mature into infectious virions, while A capsids cannot (46). By analogy with the bacteriophages, these results suggest that B capsids represent procapsids which are intermediates in the packaging process. However, a new intermediate in the assembly process has recently been identified (41, 62). These newly identified capsid forms observed in in vitro assembly extracts have the same protein content as B capsids but are more spherical; these capsids are unstable and adopt the more angular form characteristic of B capsids after prolonged incubation in vitro. These results suggest that the unstable spherical forms may represent the true procapsid intermediate (41, 62).In many bacteriophages, the procapsid contains at least three essential components: an icosahedrally arranged protein shell, an internal scaffold, and a dodecameric ring called the portal vertex through or around which the phage DNA is taken up (8, 11, 18). For HSV-1, the outer shell is composed of four proteins: the major capsid protein, VP5; a small protein bound to hexons, VP26; and a triplex structure made up of heterotrimers of VP19C and VP23 (reviewed in reference 56). VP24, VP21, and VP22a are found in the interior of the capsid and are encoded by overlapping genes UL26 and UL26.5; VP21 and VP22a are present in B but not A or C capsids and are considered to make up the internal scaffold (reviewed in reference 56). Although bacteriophages contain a portal vertex, no such structure has been observed in HSV-1 capsids. Whether the herpesviruses have a unique portal vertex through which viral DNA is taken up is unclear; it is possible that this type of unique vertex is only needed in viruses which have a tail. Capsids indistinguishable from those isolated from HSV-1-infected cells have been observed in extracts from insect cells infected with recombinant baculoviruses bearing HSV-1 capsid genes (42, 60). Therefore, it is clear that these proteins are sufficient for capsid assembly in vitro; however, it is not known whether capsids formed in vitro are competent for DNA uptake. It is possible that minor components of capsids play important roles in genome encapsidation.In addition to the capsid proteins, at least six genes are essential for the encapsidation of viral DNA: the UL6, UL15, UL25, UL28, UL32, and UL33 genes. Temperature-sensitive (ts) strains with mutations in these genes have similar phenotypes, in that viral DNA can be replicated but not cleaved and packaged (1, 2, 4, 6, 48, 51, 54, 55, 66). Strains with null mutations in the UL6, UL15, UL25, UL28, and UL33 genes have been isolated and characterized, thereby confirming the roles of these genes in cleavage and packaging (5, 27, 37, 45, 59, 68). Despite the identification of these required genes, the mechanism by which viral DNA is cleaved and packaged is not understood, nor has the role of any of the gene products been determined. The UL6 and UL25 proteins have been detected in A, B, and C capsids as well as in virions (3, 28, 37, 44); however, the precise role of these two proteins in capsids remains to be determined.A ts UL32 mutant, tsN20, defective in cleavage and packaging, has been reported previously (51). Because mutants with lesions resulting in temperature sensitivity are often prone to problems associated with incomplete penetrance at the nonpermissive temperature, we isolated a UL32 insertion mutant, hr64. Characterization of hr64 confirms that UL32 is essential for cleavage and packaging. Previous studies demonstrated that UL32 localizes to the cytoplasm of infected cells (13). We have used a functional epitope-tagged version of UL32 to confirm that in infected cells, this protein is mainly cytoplasmic, although some nuclear staining was observed.HSV-1 DNA replication occurs in globular nuclear domains termed “replication compartments” initially identified by ICP8 (UL29) staining patterns in an immunofluorescence assay (49). All seven replication proteins have now been localized within replication compartments (10, 24, 29–31, 43) as has regulatory protein ICP4 (26, 50). Ward et al. have recently reported that at late times after infection (18 h), capsids accumulate in the nucleus in regions distinct from replication compartments (64). These authors suggest that these regions represent assembly stations in which DNA is packaged. We report herein, however, that at 6 and 8 h postinfection, capsids colocalize with ICP8 in replication compartments. This suggests that at these early times, cleavage and packaging occur within replication compartments. Furthermore, we report that in cells infected with the UL32 mutant virus, capsids are distributed throughout the nucleus, accumulating in regions outside the replication compartments. This suggests that UL32 may play a role in the efficient localization of capsids in infected cells.     

Bloom Syndrome Helicase Stimulates RAD51 DNA Strand Exchange Activity through a Novel Mechanism

Loss or inactivation of BLM, a helicase of the RecQ family, causes Bloom syndrome, a genetic disorder with a strong predisposition to cancer. Although the precise function of BLM remains unknown, genetic data has implicated BLM in the process of genetic recombination and DNA repair. Previously, we demonstrated that BLM can disrupt the RAD51-single-stranded DNA filament that promotes the initial steps of homologous recombination. However, this disruption occurs only if RAD51 is present in an inactive ADP-bound form. Here, we investigate interactions of BLM with the active ATP-bound form of the RAD51-single-stranded DNA filament. Surprisingly, we found that BLM stimulates DNA strand exchange activity of RAD51. In contrast to the helicase activity of BLM, this stimulation does not require ATP hydrolysis. These data suggest a novel BLM function that is stimulation of the RAD51 DNA pairing. Our results demonstrate the important role of the RAD51 nucleoprotein filament conformation in stimulation of DNA pairing by BLM.Mutations of BLM helicase cause Bloom syndrome (BS),² a rare autosomal disorder, which is associated with stunted growth, facial sun sensitivity, immunodeficiency, fertility defects, and a greatly elevated incidence of many types of cancer occurring at an early age (1). BLM belongs to the highly conserved family of RecQ helicases that are required for the maintenance of genome integrity in all organisms (2, 3). There are five RecQ helicases in humans; mutations in three of them, WRN, RECQ4, and BLM, have been associated with the genetic abnormalities known as Werner, Rothmund-Thomson, and Bloom syndrome, respectively (4, 5). The cells from BS patients display genomic instability; the hallmark of BS is an increase in the frequency of sister chromatid and interhomolog exchanges (1, 6). Because homologous recombination (HR) is responsible for chromosomal exchanges, it is thought that BLM helicase functions in regulating HR (7–9). Also, BLM helicase is required for faithful chromosome segregation (10) and repair of stalled replication forks (11, 12), the processes that are linked to HR (13–15). BLM was found to interact physically with RAD51, a key protein of HR (16) that catalyzes the central steps in HR including the search for homology and the exchange of strands between homologous ssDNA and dsDNA sequences (17). In cells, BLM forms nuclear foci, a subset of which co-localize with RAD51. Interestingly, the extent of RAD51 and BLM co-localization increases in response to ionizing radiation, indicating a possible role of BLM in the repair of DNA double-strand breaks (16).Biochemical studies suggest that BLM may perform several different functions in HR. BLM was shown to promote the dissociation of HR intermediates (D-loops) (18–20), branch migration of Holliday junctions (21), and dissolution of double Holliday junctions acting in a complex with TopoIIIα and BLAP75 (22–24). BLM may also facilitate DNA synthesis during the repair process by unwinding the DNA template in front of the replication fork (25). In addition, BLM and its yeast homolog Sgs1 may play a role at the initial steps of DNA double-strand break repair by participating in exonucleolitic resection of the DNA ends to generate DNA molecules with the 3′-ssDNA tails, a substrate for RAD51 binding (26–29).In vivo, the process of HR is tightly regulated by various mechanisms (30). Whereas some proteins promote HR (14, 31), others inhibit this process, thereby preventing its untimely initiation (32, 33). Disruption of the Rad51-ssDNA nucleoprotein filament appears to be an especially important mechanism of controlling HR. This filament disruption activity was demonstrated for the yeast Srs2 helicase (34, 35) and human RECQ5 helicase (36). Recently, we found that BLM can also catalyze disruption of the RAD51-ssDNA filament (25). This disruption only occurs if the filament is present in an inactive ADP-bound form, e.g. in the presence of Mg²⁺. Conversion of RAD51 into an active ATP-bound form, e.g. in the presence of Ca²⁺ (37), renders the filament resistant to BLM disruption (25). In this study, we analyze the interactions of BLM with an active ATP-bound RAD51-ssDNA filament. Surprisingly, we found that BLM stimulates the DNA strand exchange activity of RAD51. Thus, depending on the conformational state of the RAD51 nucleoprotein filament, BLM may either inhibit or stimulate the DNA strand exchange activity of RAD51. Our analysis demonstrated that, in contrast to several known stimulatory proteins that act by promoting formation of the RAD51-ssDNA filament, BLM stimulates the DNA strand exchange activity of RAD51 at a later stage, during synapsis. Stimulation appears to be independent of the ATPase activity of BLM. We suggest that this stimulation of RAD51 may represent a novel function of BLM in homologous recombination.     

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.