Jamip1 (marlin-1) defines a family of proteins interacting with janus kinases and microtubules

Jamip1 (Jak and microtubule interacting protein), an alias of Marlin-1, was identified for its ability to bind to the FERM (band 4.1 ezrin/radixin/moesin) homology domain of Tyk2, a member of the Janus kinase (Jak) family of non-receptor tyrosine kinases that are central elements of cytokine signaling cascades. Jamip1 belongs to a family of three genes conserved in vertebrates and is predominantly expressed in neural tissues and lymphoid organs. Jamip proteins lack known domains and are extremely rich in predicted coiled coils that mediate dimerization. In our initial characterization of Jamip1 (73 kDa), we found that it comprises an N-terminal region that targets the protein to microtubule polymers and, when overexpressed in fibroblasts, profoundly perturbs the microtubule network, inducing the formation of tight and stable bundles. Jamip1 was shown to associate with two Jak family members, Tyk2 and Jak1, in Jurkat T cells via its C-terminal region. The restricted expression of Jamip1 and its ability to associate to and modify microtubule polymers suggest a specialized function of these proteins in dynamic processes, e.g. cell polarization, segregation of signaling complexes, and vesicle traffic, some of which may involve Jak tyrosine kinases.     

A short C-terminal domain of Yku70p is essential for telomere maintenance

The Yku heterodimer from Saccharomyces cerevisiae, comprising Yku70p and Yku80p, is involved in the maintenance of a normal telomeric DNA end structure and is an essential component of nonhomologous end joining (NHEJ). To investigate the role of the Yku70p subunit in these two different pathways, we generated C-terminal deletions of the Yku70 protein and examined their ability to complement the phenotypes of a yku70(-) strain. Deleting only the 30 C-terminal amino acids of Yku70p abolishes Yku DNA binding activity and causes a yku(-) phenotype; telomeres are shortened, and NHEJ is impaired. Using conditions in which at least as much mutant protein as full-length protein is normally detectable in cell extracts, deleting only 25 C-terminal amino acids of Yku70p results in no measurable effect on DNA binding of the Yku protein, and the cells are fully proficient for NHEJ. Nevertheless, these cells display considerably shortened telomeres, and significant amounts of single-stranded overhangs of the telomeric guanosine-rich strands are observed. Co-overexpression of this protein with Yku80p could rescue some but not all of the telomere-related phenotypes. Therefore, the C-terminal domain in Yku70p defines at least one domain that is especially involved in telomere maintenance but not in NHEJ.     

Structure-Function Correlation of G6, a Novel Small Molecule Inhibitor of Jak2: INDISPENSABILITY OF THE STILBENOID CORE*

Somatic mutations in the Jak2 protein, such as V617F, cause aberrant Jak/STAT signaling and can lead to the development of myeloproliferative neoplasms. This discovery has led to the search for small molecule inhibitors that target Jak2. Using structure-based virtual screening, our group recently identified a novel small molecule inhibitor of Jak2 named G6. Here, we identified a structure-function correlation of this compound. Specifically, five derivative compounds of G6 having structural similarity to the original lead compound were obtained and analyzed for their ability to (i) inhibit Jak2-V617F-mediated cell growth, (ii) inhibit the levels of phospho-Jak2, phospho-STAT3, and phospho-STAT5; (iii) induce apoptosis in human erythroleukemia cells; and (iv) suppress pathologic cell growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Additionally, we computationally examined the interactions of these compounds with the ATP-binding pocket of the Jak2 kinase domain. We found that the stilbenoid core-containing derivatives of G6 significantly inhibited Jak2-V617F-mediated cell proliferation in a time- and dose-dependent manner. They also inhibited phosphorylation of Jak2, STAT3, and STAT5 proteins within cells, resulting in higher levels of apoptosis via the intrinsic apoptotic pathway. Finally, the stilbenoid derivatives inhibited the pathologic growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Collectively, our data demonstrate that G6 has a stilbenoid core that is indispensable for maintaining its Jak2 inhibitory potential.     

Activation of Jak2 catalytic activity requires phosphorylation of Y1007 in the kinase activation loop.

The Janus protein tyrosine kinases (Jaks) play critical roles in transducing growth and differentiation signals emanating from ligand-activated cytokine receptor complexes. The activation of the Jaks is hypothesized to occur as a consequence of auto- or transphosphorylation on tyrosine residues associated with ligand-induced aggregation of the receptor chains and the associated Jaks. In many kinases, regulation of catalytic activity by phosphorylation occurs on residues within the activation loop of the kinase domain. Within the Jak2 kinase domain, there is a region that has considerable sequence homology to the regulatory region of the insulin receptor and contains two tyrosines, Y1007 and Y1008, that are potential regulatory sites. In the studies presented here, we demonstrate that among a variety of sites, Y1007 and Y1008 are sites of trans- or autophosphorylation in vivo and in in vitro kinase reactions. Mutation of Y1007, or both Y1007 and Y1008, to phenylalanine essentially eliminated kinase activity, whereas mutation of Y1008 to phenylalanine had no detectable effect on kinase activity. The mutants were also examined for the ability to reconstitute erythropoietin signaling in gamma2 cells, which lack Jak2. Consistent with the kinase activity, mutation of Y1007 to phenylalanine eliminated the ability to restore signaling. Moreover, phosphorylation of a kinase-inactive mutant (K882E) was not detected, indicating that Jak2 activation during receptor aggregation is dependent on Jak2 and not another receptor-associated kinase. The results demonstrate the critical role of phosphorylation of Y1007 in Jak2 regulation and function.     

Fusion proteins of Hsp70 with tumor-associated antigen acting as a potent tumor vaccine and the C-terminal peptide-binding domain of Hsp70 being essential in inducing antigen-independent anti-tumor response in vivo

Hsp70s are a family of ATP-dependent chaperones of relative molecular mass around 70 kDa. Immunization of mice with Hsp70 isolated from tumor tissues has been proved to elicit specific protective immunity against the original tumor challenge. In this work, we investigated whether Hsp70 can be used as vehicle to elicit immune response to its covalence-accompanying antigen. A recombinant protein expression vector was constructed that permitted the production of recombinant protein fusing tumor-associated antigen (eg, Mela) to the C terminus of Hsp70. We found that the Hsp70-Mela fusion protein can elicit strong cellular immune responses against murine tumor B16, which expresses protein Mela. The Hsp70 peptide-binding domain deletion mutant of the fusion protein was sufficient for inducing Mela-specific cytotoxic T lymphocyte but was not sufficient for engendering potent anti-tumor immunity against B16. We also found that host natural killer (NK) cells were stimulated in vivo by C-terminal domain of Hsp70. We thus presume that Hsp70 fusion proteins suppress tumor growth via at least 2 distinct pathways: one is covalence-accompanying antigen dependent; another is antigen independent. The C-terminal domain of Hsp70 seemed to be the crucial part in eliciting antigen-independent responses, including NK cell stimulation, against tumor challenges. Furthermore, we found that immunization with multiple Hsp70 fusion proteins resulted in a better anti-tumor effect.     

Janus kinase 2 activation by the platelet-activating factor receptor (PAFR): roles of Tyk2 and PAFR C terminus

Platelet-activating factor (PAF) is a phospholipid with multiple physiological and pathological actions. The PAF receptor (PAFR) belongs to the G protein-coupled, heptahelical receptor superfamily. Recently, we have shown that PAF signals through the Janus kinase (Jak)/STAT pathway and that Tyk2 plays an essential role in PAF-induced PAFR promoter 1 activation. In the present study we found that PAF stimulated Jak2 tyrosine phosphorylation in the monocytic cell line MonoMac-1 as well as in COS-7 cells transfected with PAFR and Jak2 cDNAs. The use of a G protein-uncoupled PAFR (D289A) mutant indicated that Jak2 activation was G protein independent. Interestingly, following PAF stimulation, Jak2 coimmunoprecipitated with PAFR in the presence of active Tyk2, but not with a kinase-inactive Tyk2 mutant, K930I. Moreover, Tyk2-K930I completely blocked PAF-stimulated Jak2 phosphorylation. Gradual deletion of C-terminal residues of the PAFR resulted in progressively decreased Jak2 activation. Deletion of 12 C-terminal residues in mutant V330Stop diminished Jak2 tyrosine phosphorylation by 17%. Further deletions of 25-37 residues from the PAFR C-tail (C317Stop, M311Stop, and T305Stop) resulted in a 50% decrease in Jak2 phosphorylation compared with the wild-type receptor. Complete removal of the C tail resulted in a mutant (K298Stop) that failed to activate Jak2, suggesting that the receptor C-terminal region contains important domains for Jak2 activation. Finally, the coexpression of a minigene encoding the C terminus of PAFR partially inhibited PAF-induced kinase activation. Taken together, our results indicate that PAF activates Jak2 and that Tyk2 and the C-terminal tail of PAFR are of critical importance for PAF-induced Jak2 activation.     

The mechanism of bacterial infection by filamentous phages involves molecular interactions between TolA and phage protein 3 domains

The early events in filamentous bacteriophage infection of gram-negative bacteria are mediated by the gene 3 protein (g3p) of the virus. This protein has a sophisticated domain organization consisting of two N-terminal domains and one C-terminal domain, separated by flexible linkers. The molecular interactions between these domains and the known bacterial coreceptor protein (TolA) were studied using a biosensor technique, and we report here on interactions of the viral coat protein with TolA, as well as on interactions between the TolA molecules. We detected an interaction between the pilus binding second domain (N2) of protein 3 and the bacterial TolA. This novel interaction was found to depend on the periplasmatic domain of TolA (TolAII). Furthermore, extensive interaction was detected between TolA molecules, demonstrating that bacterial TolA has the ability to interact functionally with itself during phage infection. The kinetics of g3p binding to TolA is also different from that of bacteriocins, since both N-terminal domains of g3p were found to interact with TolA. The multiple roles for each of the separate g3p and TolA domains imply a delicate interaction network during the phage infection process and a model for the infection mechanism is hypothesized.     

The cDNA sequence for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain) reveals a multidomain protein with internal repeats

We have isolated and sequenced a full-length cDNA clone for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain). This sequence predicts a 339 amino acid (Mr 38,493) protein containing an N-terminal region of 20 amino acids, known to interact with a 10 kd protein (light chain), and a C-terminal region, found to contain two Ca2+/phospholipid-binding sites, that can be aligned as four 70 amino acid repeats. A single p36 gene was detected in the mouse genome, and a major p36 mRNA of 1.6 kb was found to be expressed in different mouse tissues. Unexpectedly, p36 and the phospholipase A2 inhibitor lipocortin I were found to be 50% identical in sequence over the C-terminal 300 residues. The function of p36 and its relation to other proteins are discussed.     

Adapter Protein Shc Regulates Janus Kinase 3 Phosphorylation

Although constitutive activation of Janus kinase 3 (Jak3) leads to different cancers, the mechanism of trans-molecular regulation of Jak3 activation is not known. Previously we reported that Jak3 interactions with adapter protein p52ShcA (Shc) facilitate mucosal homeostasis. In this study, we characterize the structural determinants that regulate the interactions between Jak3 and Shc and demonstrate the trans-molecular mechanism of regulation of Jak3 activation by Shc. We show that Jak3 autophosphorylation was the rate-limiting step during Jak3 trans-phosphorylation of Shc where Jak3 directly phosphorylated two tyrosine residues in Src homology 2 (SH2) domain and one tyrosine residue each in calponin homology 1 (CH1) domain and phosphotyrosine interaction domain (PID) of Shc. Direct interactions between mutants of Jak3 and Shc showed that although FERM domain of Jak3 was sufficient for binding to Shc, CH1 and PID domains of Shc were responsible for binding to Jak3. Functionally Jak3 was autophosphorylated under IL-2 stimulation in epithelial cells. However, Shc recruited tyrosine phosphatases SHP2 and PTP1B to Jak3 and thereby dephosphorylated Jak3. Thus we not only characterize Jak3 interaction with Shc, but also demonstrate the molecular mechanism of intracellular regulation of Jak3 activation where Jak3 interactions with Shc acted as regulators of Jak3 dephosphorylation through direct interactions of Shc with both Jak3 and tyrosine phosphatases.     

Possible involvement of a novel STAM-associated molecule "AMSH" in intracellular signal transduction mediated by cytokines.

STAM containing an SH3 (Src homology 3) domain and an immunoreceptor tyrosine-based activation motif was previously revealed to be implicated in signaling pathways immediately downstream of Jak2 and Jak3 tyrosine kinases associated with cytokine receptors. We molecularly cloned a novel molecule interacting with the SH3 domain of STAM, which was named AMSH (associated molecule with the SH3 domain of STAM). AMSH contains a putative bipartite nuclear localization signal and a homologous region of a c-Jun activation domain-binding protein 1 (JAB1) subdomain in addition to a binding site for the SH3 domain of STAM. AMSH mutant deleted of the C-terminal half conferred dominant negative effects on signaling for DNA synthesis and c-myc induction mediated by interleukin 2 and granulocyte macrophage-colony-stimulating factor. These results suggest that AMSH plays a critical role in the cytokine-mediated intracellular signal transduction downstream of the Jak2/Jak3.STAM complex.     

Characterization of oligomeric species in the fibrillization pathway of the yeast prion Ure2p

The [URE3] prion of Saccharomyces cerevisiae shares many features with mammalian prions and poly-glutamine related disorders and has become a model for studying amyloid diseases. The development of the [URE3] phenotype is thought to be caused by a structural switch in the Ure2p protein. In [URE3] cells, Ure2p is found predominantly in an aggregated state, while it is a soluble dimer in wild-type cells. In vitro, Ure2p forms fibrils with amyloid-like properties. Several studies suggest that the N-terminal domain of Ure2p is essential for prion formation. In this work, we investigated the fibril formation of Ure2p by isolating soluble oligomeric species, which are generated during fibrillization, and characterized them with respect to size and structure. Our data support the critical role of the N-terminal domain for fibril formation, as we observed fibrils in the presence of 5 M guanidinium chloride, conditions at which the C-terminal domain is completely unfolded. Based on fluorescence measurements, we conclude that the structure of the C-terminal domain is very similar in dimeric and fibrillar Ure2p. When studying the time course of fibrillization, we detected the formation of small, soluble oligomeric species during the early stages of the process. Their remarkable resistance against denaturants, their increased content of beta-structure, and their ability to 'seed' Ure2p fibrillization suggest that conversion to the amyloid-like conformation has already occurred. Thus, they likely represent critical intermediates in the fibrillization pathway of Ure2p.     

HEDJ, an Hsp40 co-chaperone localized to the endoplasmic reticulum of human cells

Hsp40 co-chaperones, characterized by the presence of a highly conserved J domain, are involved in nearly all aspects of protein synthesis, folding, and secretion. Within the lumen of the endoplasmic reticulum, these chaperones are also involved in reverse translocation and degradation of misfolded proteins. We describe here the cloning and characterization of a novel Hsp40 chaperone, which we named HEDJ. Epitope-tagged HEDJ was demonstrated by confocal microscopy to be localized to the endoplasmic reticulum. Protease susceptibility, glycosidase treatment, and detergent solubility assays demonstrated that the molecule was luminally oriented and membrane-associated. In vitro experiments demonstrated that the J domain interacted with the endoplasmic reticulum-associated Hsp70, Bip, in an ATP-dependent manner and was capable of stimulating its ATPase activity. HEDJ mRNA expression was detected in all human tissues examined. Highly homologous sequences were found in mouse, Drosophila, and Caenorhabditis elegans data bases. These results suggest potential roles for HEDJ in protein import, folding, or translocation within the endoplasmic reticulum.     

Leukemia-associated mutations in SHIP1 inhibit its enzymatic activity, interaction with the GM-CSF receptor and Grb2, and its ability to inactivate PI3K/AKT signaling

The inositol 5-phosphatase SHIP1 is a negative regulator of the PI3K/AKT pathway, which is constitutively activated in 50-70% of acute myeloid leukemias (AML). Ten different missense mutations in SHIP1 have been described in 3% of AML patients suggesting a functional role of SHIP1 in AML. Here, we report the identification of two new SHIP1 mutations T162P and R225W that were detected in 2 and 1 out of 96 AML patients, respectively. The functional analysis of all 12 AML-associated SHIP1 mutations, one ALL-associated SHIP1 mutation (Q1076X) and a missense SNP (H1168Y) revealed that two mutations i.e. Y643H and P1039S abrogated the ability of SHIP1 to reduce constitutive PI3K/AKT signaling in Jurkat cells. The loss of function of SHIP1 mutant Y643H which is localized in the inositol phosphatase domain was due to a reduction of the specific activity by 84%. Because all other SHIP1 mutants had a normal enzymatic activity, we assumed that these SHIP1 mutants may be functionally impaired due to a loss of interaction with plasma membrane receptors or adapter proteins. In agreement with this model, we found that the SHIP1 mutant F28L located in the FLVR motif of the SH2 domain was incapable of binding tyrosine-phosphorylated proteins including the GM-CSF receptor and that the SHIP1 mutant Q1076X lost its ability to bind to the C-terminal SH3 domain of the adapter protein Grb2. In addition, SHIP1 mutant P1039S which does not reduce PI3K/AKT signaling anymore is located in a PXXP SH3 domain consensus binding motif suggesting that mutation of the conserved proline residue interferes with binding of SHIP1 to a so far unidentified SH3 domain containing protein. In summary, our data indicate that SHIP1 mutations detected in human leukemia patients impair the negative regulatory function of SHIP1 on PI3K/AKT signaling in leukemia cells either directly by reduced enzymatic activity or indirectly by disturbed protein interaction with tyrosine-phosphorylated membrane receptors or adapter proteins. These results further support a functional role of SHIP1 as tumor suppressor protein in the pathogenesis of AML.     

Dissection of Swa2p/auxilin domain requirements for cochaperoning Hsp70 clathrin-uncoating activity in vivo

The auxilin family of J-domain proteins load Hsp70 onto clathrin-coated vesicles (CCVs) to drive uncoating. In vitro, auxilin function requires its ability to bind clathrin and stimulate Hsp70 ATPase activity via its J-domain. To test these requirements in vivo, we performed a mutational analysis of Swa2p, the yeast auxilin ortholog. Swa2p is a modular protein with three N-terminal clathrin-binding (CB) motifs, a ubiquitin association (UBA) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal J-domain. In vitro, clathrin binding is mediated by multiple weak interactions, but a Swa2p truncation lacking two CB motifs and the UBA domain retains nearly full function in vivo. Deletion of all CB motifs strongly abrogates clathrin disassembly but does not eliminate Swa2p function in vivo. Surprisingly, mutation of the invariant HPD motif within the J-domain to AAA only partially affects Swa2p function. Similarly, a TPR point mutation (G388R) causes a modest phenotype. However, Swa2p function is abolished when these TPR and J mutations are combined. The TPR and J-domains are not functionally redundant because deletion of either domain renders Swa2p nonfunctional. These data suggest that the TPR and J-domains collaborate in a bipartite interaction with Hsp70 to regulate its activity in clathrin disassembly.     

Differential requirements of gag and gamma-actin domains for transforming potential of Gardner-Rasheed feline sarcoma virus.

The oncogene of Gardner-Rasheed feline sarcoma virus (GR-FeSV) encodes the 70-kilodalton protein containing gag(p15), gamma-actin, and fgr domains. To determine the role of these domains in the biological activity of P70gag-actin-fgr, we have constructed in-frame deletion and insertion mutants of GR-FeSV. We found, first, that the gamma-actin region could be deleted without affecting the transforming ability of these constructs, although an insertion mutant in the middle of the gamma-actin domain (map position 671) was partially defective in transformation and specifically had a reduced level of in vitro autophosphorylation activity. Second, mutations affecting the C-terminal third of the gag region appeared to abolish the ability to transform NIH 3T3 cells and autophosphorylation activity. These results suggest that the gamma-actin domain is not essentially required for the transforming activity of GR-FeSV but that it may take part in maintaining the conformational integrity of P70gag-actin-fgr and that the gag(p15) domain might have a critical role in modulating the function of P70gag-actin-fgr.