Prokaryotic expression cloning of a novel human tyrosine kinase.

In our database searches, we have identified mammalian homologues of yeast actin-binding protein, twinfilin. Previous studies suggested that these mammalian proteins were tyrosine kinases, and therefore they were named A6 protein tyrosine kinase. In contrast to these earlier studies, we did not find any tyrosine kinase activity in our recombinant protein. However, biochemical analysis showed that mouse A6/twinfilin forms a complex with actin monomer and prevents actin filament assembly in vitro. A6/twinfilin mRNA is expressed in most adult tissues but not in skeletal muscle and spleen. In mouse cells, A6/twinfilin protein is concentrated to the areas at the cell cortex which overlap with G-actin-rich actin structures. A6/twinfilin also colocalizes with the activated forms of small GTPases Rac1 and Cdc42 to membrane ruffles and to cell-cell contacts, respectively. Furthermore, expression of the activated Rac1(V12) in NIH 3T3 cells leads to an increased A6/twinfilin localization to nucleus and cell cortex, whereas a dominant negative form of Rac1(V12,N17) induces A6/ twinfilin localization to cytoplasm. Taken together, these studies show that mouse A6/twinfilin is an actin monomer-binding protein whose localization to cortical G-actin-rich structures may be regulated by the small GTPase Rac1.Actin is a ubiquitous protein, being found in all eukaryotic species and cell types. In muscle cells, actin filaments are stable and organized into highly ordered structures, sarcomeres. These actin filament structures, together with myosin filaments, form the framework for muscle contraction. In other cell types, actin filaments are often very dynamic and form a number of different structures called the actin cytoskeleton. In nonmuscle cells, the actin cytoskeleton is involved in multiple cell biological processes including cell morphogenesis, motility, endo-and exocytosis, and intracellular signal transduction (30).To regulate the structure and dynamics of the actin cytoskeleton, a number of actin-binding proteins have evolved in eukaryotic organisms. These proteins can specifically interact with actin monomers or actin filaments to regulate the structural and biochemical properties of actin monomers and/or filaments. From the recent increase in sequence and structural data, it has become evident that a large number of actinbinding proteins are composed of a relatively small number of protein modules that interact with actin in a specific manner (25, 32). These protein modules include motifs that specifically interact with actin monomers (thymosin-␤-4 repeat) as well as those that interact only with actin filaments (CH domain) (32).An important class of actin-binding modules is the ADF (actin-depolymerizing factor) homology (ADF-H) domain, which has been found in three functionally distinct classes of actin-binding proteins so far. The ADF-H domain is a 15-to 20-kDa protein module that is able to interact both with actin monomers and actin filaments (15). This domain was originally found in cofilin/ADF proteins, which are co...

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“…1). However, previous biochemical studies with a human homologue of twinfilin suggested that it was a tyrosine kinase (4).…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, previous studies with these human and mouse proteins did not reveal any actinrelated activities in these proteins but instead suggested that they would constitute a novel class of protein tyrosine kinases. Therefore, these proteins were named A6 tyrosine kinases (4,5).…”

mentioning

confidence: 99%

Mouse A6/Twinfilin Is an Actin Monomer-Binding Protein That Localizes to the Regions of Rapid Actin Dynamics

Vartiainen

Ojala

Auvinen

et al. 2000

Molecular and Cellular Biology

108

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“…Phosphate groups can lead to poor cell penetration and predicted instability due to endogenous phosphatases. Because bacteria lack tyrosine kinase activity, the peptides on the phage are not expected to contain phosphorylated tyrosine residues (69). Additional evidence that the peptide phage are not phosphorylated is provided by the fact that the synthetic peptides were not phosphorylated and yet were able to effectively compete with the peptide displayed on the phage for binding to Grb7.…”

Section: Figmentioning

confidence: 99%

Identification of Novel Non-phosphorylated Ligands, Which Bind Selectively to the SH2 Domain of Grb7

Pero

Oligino

Daly

et al. 2002

Journal of Biological Chemistry

121

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Grb7 is an adapter-type signaling protein, which is recruited via its SH2 domain to a variety of receptor tyrosine kinases (RTKs), including ErbB2 and ErbB3. It is overexpressed in breast, esophageal, and gastric cancers, and may contribute to the invasive potential of cancer cells. Molecular interactions involving Grb7 therefore provide attractive targets for therapeutic intervention. We have utilized phage display random peptide libraries as a source of small peptide ligands to the SH2 domain of Grb7. Screening these libraries against purified Grb7 SH2 resulted in the identification of Grb7-binding peptide phage clones that contained a nonphosphorylated Tyr-X-Asn (YXN) motif. The tyrosinephosphorylated form of this motif is characteristic of Grb7 SH2 domain binding sites identified in RTKs and other signaling proteins such as Shc. Peptides that are non-phosphorylated have greater potential in the development of therapeutics because of the instability of a phosphate group in vivo. Using a biased library approach with this conserved YXN motif, we identified seven different peptide phage clones, which bind specifically to the SH2 domain of Grb7. These peptides did not bind to the SH2 domain of Grb2 (which also selects for Asn at pY ؉2 ) or Grb14, a closely related family member. The cyclic structure of the peptides was required to bind to the Grb7 SH2 domain. Importantly, the synthetic Grb7-binding peptide G7-18 in cell lysates was able to specifically inhibit the association of Grb7 with the ErbB family of RTKs, in particular ErbB3, in a dose-dependent manner. These peptides will be useful in the development of targeted molecular therapeutics for cancers overexpressing Grb7 and in the development of Grb7-specific inhibitors to gain a complete understanding of the physiological role of Grb7.

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“…Subdomain VIII appears to play a major role in the recognition of peptide substrates, and contains several autophosphorylation sites, required for maximal kinase activity in many protein kinases. No clear ATP-binding site is identified as in the Bcr protein serine/threonine kinase (42) and in the human A6 tyrosine kinase (43). Clone 5 could represent a new example of these atypical kinases, and work is currently in progress to test this hypothesis.…”

Section: Discussionmentioning

confidence: 99%

Identification and Characterization of Novel Genes Modulated in the Thyroid of Dogs Treated with Methimazole and Propylthiouracil

Wilkin¹,

Savonet²,

Radulescu³

et al. 1996

Journal of Biological Chemistry

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Induction of cell proliferation by mitogen or growth factor stimulation leads to the specific stimulation or repression of a large number of genes. To better understand differentiated epithelial cell growth regulation, we have initiated a study to identify genes which are regulated by the thyrotropin-dependent mitogenic pathway in dog thyroid cells. A thyroid cDNA library was prepared from a methimazole and propylthiouraciltreated dog and differentially screened with probes derived from control or stimulated thyroids. Among 19 clones isolated, 6 encode known proteins (inwardly rectifying potassium channel, nucleosome assembly protein, ribosomal protein L7, elongation factor 1␣, nonmuscle myosin light chain, and heat shock protein 90␤). The 13 others correspond to proteins whose function is unknown. Among them, 5 correspond to mRNAs whose expression was modulated by mitogenic stimulation of thyrocytes in primary culture. A preliminary characterization of two of these cDNAs is reported: clone 5, which might represent a novel, atypical protein kinase, and clone 3, which contains ankyrin-like repeats, suggesting that it might interact with other proteins.Induction of cell proliferation by mitogen or growth factor stimulation leads to the specific and sequential expression of a large number of genes (1-8): immediate early genes, induced rapidly and independently of de novo protein synthesis, delayed early genes whose transcriptional activation requires new protein synthesis, and others ending with the late G 1 genes, which are mainly enzymes involved in DNA synthesis. Conversely, the expression of other genes is also sequentially repressed after mitogenic stimulation (9 -11). Many of these regulated genes are proto-oncogenes or anti-oncogenes. Most of the known ones belong to two major classes of mitogenic pathways: the growth factors receptors tyrosine protein kinases and the phorbol ester protein kinases C cascades. A third signaling pathway, the cyclic AMP-dependent cascade, considered for a long time to be a general inhibitor of proliferation, stimulates proliferation in several epithelial cell types and in yeast (reviewed in Ref. 12). In thyroid cells, this pathway is activated by thyrotropin (TSH), 1 which is the main physiological agent regulating the thyroid gland (13,14). TSH and cyclic AMP promote cell proliferation, function, and differentiation while the mitogenic pathways elicited by EGF and tumor promoting phorbol esters are associated with the loss of expression of the differentiation specific genes (15-19). Several steps of the latter cascades are missing in the cyclic AMP pathway (14). The molecular mechanisms of this pathway are still largely unknown. We have therefore initiated a study aimed at the identification of genes that are regulated by cyclic AMP in thyroid cells.To identify new genes that might be regulated by the thyroid mitogenic pathways, we prepared a cDNA library from the thyroid of a dog treated in vivo with methimazole and propylthiouracil. The inhibition by these drugs of thyroid h...

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Prokaryotic expression cloning of a novel human tyrosine kinase.

Cited by 41 publications

References 29 publications

Mouse A6/Twinfilin Is an Actin Monomer-Binding Protein That Localizes to the Regions of Rapid Actin Dynamics

Mouse A6/Twinfilin Is an Actin Monomer-Binding Protein That Localizes to the Regions of Rapid Actin Dynamics

Identification of Novel Non-phosphorylated Ligands, Which Bind Selectively to the SH2 Domain of Grb7

Identification and Characterization of Novel Genes Modulated in the Thyroid of Dogs Treated with Methimazole and Propylthiouracil

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