Cdc37 engages in stable,S14A mutation-reinforced association with the most atypical member of the yeast kinome,Cdk-activating kinase (Cak1)

In most eukaryotes, Cdc37 is an essential chaperone, transiently associating with newly synthesised protein kinases in order to promote their stabilisation and activation. To determine whether the yeast Cdc37 participates in any stable protein interactions in vivo, genomic two-hybrid screens were conducted using baits that are functional as they preserve the integrity of the conserved N-terminal region of Cdc37, namely a Cdc37-Gal4 DNA binding domain (BD) fusion in both its wild type and its S14 nonphosphorylatable (Cdc37(S14A)) mutant forms. While this failed to identify the protein kinases previously identified as Cdc37 interactors in pull-down experiments, it did reveal Cdc37 engaging in a stable association with the most atypical member of the yeast kinome, cyclin-dependent kinase (Cdk1)-activating kinase (Cak1). Phosphorylation of the conserved S14 of Cdc37 is normally crucial for the interaction with, and stabilisation of, those protein kinase targets of Cdc37, Cak1 is unusual in that the lack of this Cdc37 S14 phosphorylation both reinforces Cak1:Cdc37 interaction and does not compromise Cak1 expression in vivo. Thus, this is the first Cdc37 client kinase found to be excluded from S14 phosphorylation-dependent interaction. The unusual stability of this Cak1:Cdc37 association may partly reflect unique structural features of the fungal Cak1.     

Protein-protein interaction of FHL2, a LIM domain protein preferentially expressed in human heart, with hCDC47

In the yeast two-hybrid library screening, the heart-specific FHL2 protein was found to interact with hCDC47. In vitro interaction study between FHL2 protein and hCDC47 was demonstrated. From the results of domain studies by the yeast two-hybrid assay, the second and third LIM domains in conjunction with the first half LIM domain of FHL2 were identified to be important in binding with hCDC47. Besides, in Northern blot hybridization of human cancer cell lines, the highest FHL2 mRNA expression was detected in colorectal adenocarcinoma SW480 and HeLa cell S3. Our results imply that FHL2 protein may associate with cancer development and may act as a molecular adapter to form a multicomplex with hCDC47 in the nucleus, thus it plays an important role in the specification or maintenance of the terminal differentiated phenotype of heart muscle cells.     

酵母双杂交筛选与蛋白激酶CK2α′相互作用蛋白——泛素-52氨基酸融合蛋白的鉴定

蛋白激酶CK2是一种真核细胞中普遍存在的信使非依赖性丝/苏氨酸蛋白激酶. 为研究CK2α′亚基在精子发生中的作用机制,将构建于pACT2质粒的人睾丸cDNA文库和人蛋白激酶CK2α′为诱饵蛋白进行酵母双杂交实验. 以初步筛选与人蛋白激酶CK2α′相互作用蛋白的阳性候选克隆,筛选获得8个阳性克隆,其中1个与人泛素-52氨基酸融合蛋白基因（UBA52）的cDNA序列有高度同源性（100％）. GST pull-down实验在细胞外进一步证实了CK2α′与UBA52之间存在相互作用. 本实验证明,人泛素-52氨基酸（UBA52）融合蛋白是人CK2α′亚基的相互作用蛋白, 它们之间的相互作用对精子发生机制的影响尚不清楚,进一步分子机制研究正在进行中.     

In vivo and in vitro association of 14-3-3 epsilon isoform with calmodulin: implication for signal transduction and cell proliferation

Using a yeast two-hybrid screen, human 14-3-3 epsilon protein was found to interact with human calmodulin. In vitro binding assay between human 14-3-3 epsilon protein/peptide and calmodulin was demonstrated by native gel electrophoresis, and the interaction was shown to be calcium dependent. Our results, along with the association of the 14-3-3 epsilon protein with other signaling proteins, suggest that the 14-3-3 protein could provide a link between signal transduction and cell proliferation.     

A novel proteasome interacting protein recruits the deubiquitinating enzyme UCH37 to 26S proteasomes

The 26S proteasome is a multisubunit protease responsible for regulated proteolysis in eukaryotic cells. It is composed of one catalytic 20S proteasome and two 19S regulatory particles attached on both ends of 20S proteasomes. Here, we describe the identification of Adrm1 as a novel proteasome interacting protein in mammalian cells. Although the overall sequence of Adrm1 has weak homology with the yeast Rpn13, the amino- and carboxyl-terminal regions exhibit significant homology. Therefore, we designated it as hRpn13. hRpn13 interacts with a base subunit Rpn2 via its amino-terminus. The majority of 26S proteasomes contain hRpn13, but a portion of them does not, indicating that hRpn13 is not an integral subunit. Intriguingly, we found that hRpn13 recruits UCH37, a deubiquitinating enzyme known to associate with 26 proteasomes. The carboxyl-terminal regions containing KEKE motifs of both hRpn13 and UCH37 are involved in their physical interaction. Knockdown of hRpn13 caused no obvious proteolytic defect but loss of UCH37 proteins and decrease in deubiquitinating activity of 26S proteasomes. Our results indicate that hRpn13 is essential for the activity of UCH37.     

Regulation of Dyrk1A kinase activity by 14-3-3

Dual-specificity tyrosine(Y) regulated kinase 1A (DYRK1A) is a serine/threonine protein kinase implicated in mental retardation resulting from Down syndrome. In this study, we carried out yeast two-hybrid screening to find proteins regulating DYRK1A kinase activity. We identified 14-3-3 as a Dyrk1A interacting protein, which is consistent with the previous finding of the interaction between the yeast orthologues Yak1p and Bmh1/2p. We showed the interaction between Dyrk1A and 14-3-3 in vitro and in vivo. The binding required the N-terminus of Dyrk1A and was independent of the Dyrk1A phosphorylation status. Functionally, 14-3-3 binding increased Dyrk1A kinase activity in a dose dependent manner in vitro. In vivo, a small peptide inhibiting 14-3-3 binding, sc138, decreased Dyrk1A kinase activity in COS7. In summary, these results suggest that DYRK1A kinase activity could be regulated by the interaction of 14-3-3.     

The 32-kilodalton subunit of replication protein A interacts with menin,the product of the MEN1 tumor suppressor gene

Menin is a 70-kDa protein encoded by MEN1, the tumor suppressor gene disrupted in multiple endocrine neoplasia type 1. In a yeast two-hybrid system based on reconstitution of Ras signaling, menin was found to interact with the 32-kDa subunit (RPA2) of replication protein A (RPA), a heterotrimeric protein required for DNA replication, recombination, and repair. The menin-RPA2 interaction was confirmed in a conventional yeast two-hybrid system and by direct interaction between purified proteins. Menin-RPA2 binding was inhibited by a number of menin missense mutations found in individuals with multiple endocrine neoplasia type 1, and the interacting regions were mapped to the N-terminal portion of menin and amino acids 43 to 171 of RPA2. This region of RPA2 contains a weak single-stranded DNA-binding domain, but menin had no detectable effect on RPA-DNA binding in vitro. Menin bound preferentially in vitro to free RPA2 rather than the RPA heterotrimer or a subcomplex consisting of RPA2 bound to the 14-kDa subunit (RPA3). However, the 70-kDa subunit (RPA1) was coprecipitated from HeLa cell extracts along with RPA2 by menin-specific antibodies, suggesting that menin binds to the RPA heterotrimer or a novel RPA1-RPA2-containing complex in vivo. This finding was consistent with the extensive overlap in the nuclear localization patterns of endogenous menin, RPA2, and RPA1 observed by immunofluorescence.     

AOP-1 interacts with cardiac-specific protein kinase TNNI3K and down-regulates its kinase activity

In the present study, a yeast two-hybrid screening system was used to identify the interaction partners of cardiac troponin I-interacting kinase (TNNI3K) that might serve as regulators or targets, and thus in turn to gain some insights on the roles of TNNI3K. After screening the adult heart cDNA library with a bait construct encoding the ANK motif of TNNI3K, antioxidant protein 1 (AOP-1) was isolated. The interaction between TNNI3K and AOP-1 was confirmed by the in vitro binding assay and coexpression experiments in vivo. The colocalization of TNNI3K and AOP-1 was clarified by confocal immunofluorescence. Moreover, coexpression of AOP-1 inhibited TNNI3K kinase activity in the in vitro kinase assay.     

Evidence that the Arabidopsis Ubiquitin C-terminal Hydrolases 1 and 2 associate with the 26S proteasome and the TREX-2 complex

The 26S proteasome interacts with a number of different proteins, while the TREX-2 complex is an important component of the mRNA export machinery. In animals and yeast, members of the Ubiquitin C-terminal Hydrolase 37 (UCH37) family are found to associate with the 26S proteasome, but this has not been demonstrated in plants. The Arabidopsis UCH1 and UCH2 are orthologous to UCH37. Here, we show that UCH1 and UCH2 interact with the 26S proteasome lid subunits. In addition, the two UCHs also interact with TREX-2 components. Our data suggest that Arabidopsis UCHs may serve as a link between the 26S proteasome lid complex and the TREX-2 complex.     

RACK1 protein interacts with Helicobacter pylori VacA cytotoxin: the yeast two-hybrid approach.

The VacA toxin is the major virulence factor of Helicobacter pylori. The studies on VacA intracellular expression suggest that it interacts with cytosolic proteins and that this interaction contributes significantly to vacuolization. The aim of this study was to identify the host protein(s) that interacts with the VacA protein. We used the fragments of VacA protein fused with GAL4-BD as the baits in the yeast two-hybrid approach. The yeast transformed with plasmids encoding bait proteins were screened with human gastric mucosa cDNA library, encoded C-terminal fusion proteins with GAL4-AD. Three independent His-beta-Gal-positive clones were identified in VacA-b1 screen; they matched two different lengths of cDNA encoding RACK1 protein. The specific activity of beta-galactosidase found in the yeast expressing both VacA-b1 and RACK1 fusion proteins was 12-19 times higher compared to all negative controls used. VacA is capable of binding the RACK1 in vitro as was confirmed by the pull-down assay with GST fusion VacA protein and [(35)S]Met-labeled RACK1 protein fragments.     

Identification and characterization of the interaction between tuberin and 14-3-3zeta

Tuberous sclerosis is caused by mutations to either the TSC1 or TSC2 tumor suppressor gene. The disease is characterized by a broad phenotypic spectrum that includes seizures, mental retardation, renal dysfunction, and dermatological abnormalities. TSC1 encodes a 130-kDa protein called hamartin, and TSC2 encodes a 200-kDa protein called tuberin. Although it has been shown that hamartin and tuberin form a complex and mediate phosphoinositide 3-kinase/Akt-dependent phosphorylation of the ribosomal protein S6, it is not yet clear how inactivation of either protein leads to tuberous sclerosis. Therefore, to obtain additional insight into tuberin and hamartin function, yeast two-hybrid screening experiments were performed to identify proteins that interact with tuberin. One of the proteins identified was 14-3-3zeta, a member of the 14-3-3 protein family. The interaction between tuberin and 14-3-3zeta was confirmed in vitro and by co-immunoprecipitation; multiple sites within tuberin for 14-3-3zeta binding were identified; and it was determined that 14-3-3zeta associated with the tuberin-hamartin complex. Finally, it was shown that the tuberin/14-3-3zeta interaction is regulated by Akt-mediated phosphorylation of tuberin, providing insight into how tuberin may regulate phosphorylation of S6.     

Hepatitis C virus core protein binds to a DEAD box RNA helicase.

Approximately 4 million Americans are infected with the hepatitis C virus (HCV), making it a major cause of chronic liver disease. Because of the lack of an efficient cell culture system, little is known about the interaction between HCV and host cells. We performed a yeast two-hybrid screen of a human liver cell cDNA library with HCV core protein as bait and isolated the DEAD box protein DBX. DBX has significant amino acid sequence identity to mouse PL10, an ATP-dependent RNA helicase. The binding of DBX to HCV core protein occurred in an in vitro binding assay in the presence of 1 M NaCl or detergent. When expressed in mammalian cells, HCV core protein and DBX were co-localized at the endoplasmic reticulum. In a mutant strain of Saccharomyces cerevisiae, DBX complemented the function of Ded1p, an essential DEAD box RNA helicase. HCV core protein inhibited the growth of DBX-complemented mutant yeast but not Ded1p-expressing yeast. HCV core protein also inhibited the in vitro translation of capped but not uncapped RNA. These findings demonstrate an interaction between HCV core protein and a host cell protein involved in RNA translation and suggest a mechanism by which HCV may inhibit host cell mRNA translation.     

A novel regulatory function of selenocysteine lyase, a unique catalyst to modulate major urinary protein

Mouse selenocysteine lyase (SCL) catalyzes the decomposition of -selenocysteine into -alanine and selenium with pyridoxal 5′-phosphate as a coenzyme. When using SCL as bait in a yeast two-hybrid screening method, major urinary protein I (MUP-I) was identified as a protein that interacts with SCL. This interaction was confirmed with an in vitro binding assay. MUP-I is known as a pheromone-binding protein that accommodates volatile effectors to affect the physiology and behavior of mice. We found that the binding of 2-naphthol to MUP-I was significantly inhibited by SCL, suggesting that SCL regulates the binding capacity of MUP-I.     

Functional analysis of a novel gene, DD3-3, from Dictyostelium discoideum

Mutations in MYOC gene encoding myocilin are responsible for primary open-angle glaucoma (POAG). In order to search for protein(s) that can interact with myocilin, we screened a human skeletal muscle cDNA library using yeast two-hybrid system and identified flotillin-1, a structural protein of lipid raft that is detergent-resistant and a liquid ordered microdomain, as a protein interacting with myocilin. The interaction was confirmed by in vitro glutathione S-transferase pulldown and in vivo co-immunoprecipitation studies. In yeast two-hybrid assay, the C-terminus of myocilin, an olfactomedin-like domain in which most mutations related to POAG are scattered, was found to be necessary and sufficient for the interaction. However, myocilins with mutations such as G364V, K423E, and Y437H on the domain failed to interact with flotillin-1. Although the physiological significance of the interaction has yet to be elucidated, our results showed that the alteration of the interaction by mutations in MYOC might be a key factor of the pathogenesis of POAG.     

Direct interaction of nerve growth factor receptor, TrkA, with non-receptor tyrosine kinase, c-Abl, through the activation loop

The nerve growth factor receptor, TrkA, is essential for the survival and differentiation of neurons in the central and peripheral nervous systems. To understand the molecular principles underlying this differentiation step, we employed a yeast two-hybrid screening protocol using human TrkA as bait. We isolated c-Abl as a TrkA-interacting protein, in addition to known proteins such as phospholipase Cgamma and SH2-B. This interaction was confirmed by an in vitro binding assay using glutathione S-tranferase-Abl fusion protein. Furthermore, we show here that c-Abl binds to phosphotyrosine residue(s) in the kinase activation loop of TrkA.     

Trichosanthin interacts with acidic ribosomal proteins P0 and P1 and mitotic checkpoint protein MAD2B.

Trichosanthin is a ribosome-inactivating protein with multiple pharmacological properties. By a yeast two-hybrid system, ribosomal phosphoproteins P0 and P1 and a putative mitotic checkpoint protein, MAD2B, were found to interact with an active-site mutated trichosanthin (TCS). The interactions were verified by an in vitro binding assay of recombinant wild-type TCS and target proteins. The interaction domain of P0 was mapped to amino acids 220-273, which had been previously reported to be involved in the interaction with P1 and P2 in yeast. Consistent with our previous finding that the last seven residues of TCS are not essential for an active conformation, the same deletion did not affect the interaction with P0. Our present study suggests that TCS may disrupt the binding of elongation factors to the P-complex, in addition to the well-known N-glycosidase activity for ribosome inactivation.