SH2-containing inositol polyphosphate 5-phosphatases, SHIP2, has been established as a regulator of the insulin cascade, of cell adhesion and spreading, actin structures, remodelling and cytoskeletal organization. However, the molecular mechanisms underlying these processes still needed additional investigations. Among different regulatory mechanisms, protein-protein interaction play an essential role. To better understand the molecular mechanism of SHIP2 in signalling pathway as well as to reveal novel roles of SHIP2, a two-hybrid was performed to search for SHIP2 protein interactors. JNK-interacting protein 1 (JIP1) and intersectin 1 (ITSN1) were two of the newly identified protein partners of SHIP2. In this thesis, we characterized the associations of SHIP2 with JIP1 and ITSN1 in different aspects as identifying the interacting domain involved, biochemical function regulations and cellular biological roles. The JIP scaffold family of proteins associate with MAPK, MAPKK and MAPKKK creating functional signaling modules to control the specificity of signal transduction. JIP1 is characterized as a scaffold protein assembling JNK, MAPK kinase 7 (MKK7), mixed lineage kinase (MLK), dual leucine zipper-bearing kinase (DLK). It thus enhances the selectivity and effectiveness of kinase activation during JNK signaling. In this thesis, the SHIP2-JIP1 interaction has been confirmed both in overexpression system in COS-7 and CHO-IR cells, and in native cells of COS-7. Both the proline-rich (PR) domain (residues 359-487) and PTB domain of JIP1 participated in this interaction. Overexpression of SHIP2 in COS-7 cells up-regulated JIP1-mediated JNK activation and the tyrosine phosphorylations of both JIP1 and MLK3. These effects were independent of SHIP2 catalytic activity. By the use of kinase inhibitors, we showed that Abl and Src family tyrosine kinases might be implicated in the regulation of JIP1 tyrosine phosphorylation. The residue Y270 of JIP1, a potential target of Abl tyrosine kinase, was shown to be involved in SHIP2-increased JIP1 tyrosine phosphorylation. In an in vitro assay, JIP1 negatively regulated the catalytic activity of SHIP2. In addition, upon the stimulation of okadaic acid, the overexpression of SHIP2 caused less viability of COS-7 cells. These data provide a new molecular link between SHIP2 and JIP1-mediated JNK pathway, and may help explain the biochemical mechanisms of SHIP2 in cellular apoptosis, as well as in insulin pathway. Another protein partner, ITSN1, is a multi-domain protein which plays a role in endocytosis, MAPK signalling and actin cytoskeleton. The interaction between SHIP2 and ITSN1 was confirmed in overexpression systems in COS-7 cells, as well as at the physiological concentration with the endogenously expressed proteins in C2C12 and COS-7 cells. EGF stimulation did not modulate the association of SHIP2 and ITSN1. ITSN1-SH3D, A, C and E domains interacted with the C-terminal part of SHIP2 with the binding affinity as SH3D>SH3A>SH3C>SH3E. Upon the stimulation of EGF, the expression of SHIP2 may recruit ITSN1 short form (ITSN1-S) to cell membrane. The ITSN-mediated ERK1/2 and JNK activations in response to EGF were not modulated when SHIP2 or catalytic mutant of SHIP2 or TSHIP2 was overexpressed. The link between SHIP2 and ITSN may provide one of the molecular mechanisms used by SHIP2 to participate in receptor endocytosis regulation. In conclusion, our data of the associations of SHIP2 with JIP1 and ITSN1 provide evidence for potential novel biochemical mechanisms of SHIP2 to be implicated in JNK pathway as well as EGF receptor endocytosis. JIP1 and ITSN1, which are both implicated in the JNK pathway, may also have a link through the common protein partner SHIP2, giving rise to potential interesting study goal.

This study focuses on the mechanism of transmembrane signaling by Tar, the aspartate chemoreceptor of Escherichia coli. Like other bacterial chemoreceptors, Tar localizes to the cell membrane and relays information about the external chemical environment through the membrane to a cytoplasmic signaling domain. The output of the signaling domain controls the directional bias of the rotary flagellar motors of the cell. Net movement of a cell in a chemical gradient involves temporal comparison of the current concentration with the concentration in the recent (a few seconds) past. The current concentration is measured as the percent occupancy of the extracellular ligand-binding domain of the receptor, and the past is represented by the extent of covalent methylation of four conserved glutamyl residues in the cytoplasmic domain. Under steady-state conditions, the methylation level corresponds to ligand occupancy. Tar is a dimer, and much evidence suggests that dimers associate into trimers of dimers. Higher-order arrays of receptors form in the presence of the cytoplasmic proteins CheA and CheW. The conformational change generated by ligand binding is transmitted through the membrane by one subunit of a dimer. To examine whether this initially asymmetric signal becomes symmetric within the cytoplasmic domain, I examined aspartate-induced adaptive methylation of the two subunits of mutant Tar receptor heterodimers. In the presence of CheA and CheW, adaptive methylation after addition of aspartate was symmetric, but in their absence, although the level of methylation increased, the rates were different for the two subunits. I also found that cross-talk, at the level of adaptive methylation, occurs between different receptor types even in the absence of CheA and CheW. These results provide support for the idea that a tight association of receptor dimers within trimers of dimers allows for an actively signaling receptor to affect the methylation state, and thus presumably the signaling state, of receptors within a trimer that are not bound to an attractant ligand.

Thesis (Ph. D.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 85-112).

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains x, 92 p.: ill. Includes abstract and vita. Advisor: Clay B. Marsh, Dept. of Veterinary Biosciences. Includes bibliographical references (p. 84-92).

Hedgehog (Hh) is an important morphogen that dictates tissue patterning during embryonic development. Recent studies showed that mutation in Indian Hedgehog(IHH)resulted in Brachydactyly type A1(BDA1);however, the disease pathogenesis in patients without IHH mutation remained unknown. PDZD2 is a multi-PDZ domain-containing protein of unknown function in early development. Human PDZD2 is mapped to chromosome 5p13.2, which co-localizes with the disease-associated gene in a family of BDA1 patients, suggesting involvement of PDZD2 in limb development. In situ hybridization revealed that Pdzd2 was expressed in the distal mesenchyme partially overlapping with Shh expressionin mouse limb bud. During digit patterning, Pdzd2 was expressed in the interzone regions that flanked the Ihh/Gli1-expressing phalanx condensation. Moreover,Pdzd2 was expressed in the paraxial mesoderm adjacent to the differentiating neural tube. Pdzd2expression in various Hh-active tissues in mouse and chicken suggested an evolutionary conserved role of PDZD2 in modulating general Hh signaling during early development. Interestingly, PDZD2 protein was detected at the neural tube away from its site of synthesis, suggesting a non-cell autonomous role of PDZD2 possibly via its secreted form, sPDZD2. Functional studies showed that overexpression of sPDZD2 in the chicken neural tube leads to down-regulation ofNKX2.2andOLIG2expression.sPDZD2 was shown to counteract the ectopic NKX2.2 expression induced by long-range signaling of ectopic HH. Consistently, sPDZD2exhibited an inhibitory effect on SHH-induced reporter activity in a Gli-luciferase cell line. For in vivo analysis, a transgenic mouse line carrying a floxed Pdzd2 allele (Pdzd2fl) was generated to ablate Pdzd2 expression.Pdzd2+/fl mice were crossed with Protamine-cre to generate the null allele (Pdzd2-). However, heterozygous intercross yielded no homozygous mutant as early as E9.5, suggesting early embryonic lethality. Thus, conditional Pdzd2 knock-out in the limb was pursuedusingPrx1-cre.However, no significant perturbation of Hh signaling was observed in Pdzd2fl/fl:Prx1-cre limb buds, which might be due to incomplete knock-out of Pdzd2, or functional redundancy among Hh modulators. To study the relevance of Pdzd2in the development of BDA1, Pdzd2-/fl mouse was crossed with the BDA1 mouseIhhE95K/E95Kto study the effect of reducing Pdzd2expression under defective Hh signaling. Preliminary analysis of the Pdzd2+/-, Ihh+/E95K compound mutants showed more severe phenotypes comparing with IhhE95K/E95K. These included delayed limb development and further diffusion ofGli1expression in the digits, suggestive of a direct involvement of Pdzd2in BDA1. It was speculated that Pdzd2negatively modulated Ihh signaling and restricted Hh signals from entering the interzone, which was required for normal digit patterning. Depletion of Pdzd2might result in an expansion of Ihh signaling into the interzone, leading to the BDA1phenotypessimilar to the current BDA1 disease model proposed forIhhE95Kmutation. Taken together, my study revealed the novel expression of Pdzd2in close proximity to multiple Hh-active tissues during early development and provided the first evidence that PDZD2/sPDZD2 is a negative modulator of general Hh signaling. These data strongly supported PDZD2as a disease-associated locus in the family of BDA1 patients that do not carry IHHmutations. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

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The application of the proteomic approach has facilitated efforts directed toward the mapping of the entire Arabidopsis cell wall proteome. Proteins were sequentially extracted from purified cell walls using 0.2 M CaC1(_2) followed by a urea buffer. The extracts were resolved via large format two dimensional polyacrylamide gel electrophoresis (2-D PAGE) and were visualised via Coomassie brilliant blue staining. The aim was to identify and characterise as many cell wall proteins as possible, with the hope of identifying novel cell wall proteins. Out of 325 spots visualised on the 2-D polyacrylamide gel, 144 gave a positive protein identification representing 104 different proteins. The identified proteins were divided into 3 categories. The first category included proteins that have been previously identified as plant cell wall proteins. The second category was designated to include novel cell wall proteins (hypothetical proteins) and the third category was made up of proteins, which had recognised functions, but had never hitherto been known to be secreted to the extracellular matrix. Among the identified novel cell wall proteins there were several that shared high homology with protein kinases. These proteins possessed all the characteristics of secreted polypeptides, such as the cleavable N-terminal signal peptide, and were found to lack both the transmembrane domain and the endoplasmic reticulum retention tetrapeptides (HDEL and KDEL). These observations suggested that, as in animal cells, plant cells had extracellular protein kinase activity (phosphorylation). This was supported by the recent discovery that plant cells secrete ATP to the extracellular matrix (Thomas et al., 2000). Verification of the occurrence of extracellular protein kinase activity was further strengthened by the identification of phosphorylated bona fide cell wall proteins and stress responses caused by the depletion extracellular ATP.

The fungus Stagonospora nodorum is the causal agent of leaf and glume blotch disease on wheat and is an emerging model for the study of the interaction between plants and necrotrophic fungal pathogens. Signal transduction plays a critical role during infection by allowing the pathogen to sense and appropriately respond to environmental changes. The role of signal transduction in the pathogenicity of S. nodorum was analysed by the targeted inactivation of genes encoding a Gá subunit (Gna1) and a mitogen-activated protein kinase (Mak2). Strains carrying the inactivated genes were impaired in virulence and demonstrated a host of phenotypic impairments such as abolished sporulation. Therefore, it was hypothesised that Gna1 and Mak2 regulate downstream effector molecules that are critical for pathogenic development. A 2D gel-based proteomic approach was used to compare the extracellular and intracellular proteomes of the wild-type fungus and signalling mutants for differences in protein abundance. Tandem mass spectrometry (LC-MS/MS) analysis and patternmatching against the S. nodorum genome sequence led to the identification of 26 genes from 34 differentially abundant protein spots. These genes possess probable roles in protein cycling, plant cell wall degradation, stress response, nucleotide metabolism, proteolysis, quinate and secondary metabolism. A putative short-chain dehydrogenase gene (Sch1) was identified and its expression was shown to be reduced in both signalling mutants. The transcript level of Sch1 increased during the latter period of infection coinciding with pycnidiation. Sch1 was inactivated by targeted gene deletion. Mutants were able to effectively colonise the host but asexual sporulation was dramatically reduced and pycnidial ontogeny was severely disrupted. Furthermore, the sch1 mutants showed alterations in the metabolome. GC-MS analysis identified a metabolite which accumulated in the sch1 mutants. Computational and database analyses indicated that the compound possesses a cyclic carbon backbone. Based on these findings, Sch1 may be a suitable target for fungicides that inhibit asexual sporulation and the accumulated compound may be used to design novel antifungal compounds. 2D SDS-PAGE analysis identified increased abundance of another putative short-chain dehydrogenase (Sch2) and a nitroreductase in the sch1-deleted background. It was also shown that Sch2 was regulated by Gna1.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 4 uppsatser.