Biomedical subjects

K Letwin

Publications and source records attributed to K Letwin.

10 recordsLinked to original sources

PubMed2001

Functional analyses of two newly identified PITX2 mutants reveal a novel molecular mechanism for Axenfeld-Rieger syndrome.

The specific role of PITX2 in the pathogenesis of anterior segment dysgenesis has yet to be clearly defined. We provide here new insight into PITX2 pathogenesis through mutational and functional analyses. Three PITX2 mutations were found in a screen of 38 unrelated individuals affected with anterior segment anomalies (8%). All three mutations were found among the 21 individuals affected with Axenfeld-Rieger syndrome (ARS). We have identified two novel mutations, a valine-->leucine (V45L) missense mutation at position 45 within the PITX2 homeodomain, and a seven amino acid duplication (7aaDup) of residues 6-12 of the homeodomain. DNA-binding studies of the two mutant PITX2 proteins demonstrated a <10-fold reduction in the DNA-binding activity of the V45L mutant, and a >100-fold reduction in activity of the 7aaDup mutant. Luciferase reporter assays showed a >200% increase in PITX2 transactivation activity of the V45L mutant, while the 7aaDup mutant was unable to transactivate at detectable levels. Our analyses of the V45L PITX2 mutant reveal that the DNA-binding domain of PITX2 can influence transactivation activity independently of DNA binding. Furthermore, our findings expand the hypothesis that the amount of residual PITX2 activity underlies the variable severity of ocular phenotypes that result from PITX2 mutation. For the first time, we present evidence that increased PITX2 activity may underlie the severe ARS ocular phenotype. We conclude that increased activity of one PITX2 allele may be as physiologically disruptive as a mutation that nullifies a PITX2 allele, with either condition resulting in ARS.

Abnormalities, Multiple↗

PubMed1994

Immunolocalization of the Nuk receptor tyrosine kinase suggests roles in segmental patterning of the brain and axonogenesis.

Neural kinase (Nuk) encodes a murine receptor-like tyrosine kinase belonging to the Eph/Elk/Eck family. Protein localization studies indicate that during early embryogenesis Nuk is confined to the developing nervous system, where it marks segments along the axis of the neural tube in the hindbrain (rhombomeres r2, r3 and r5) and specific morphological bulges of the midbrain and forebrain. Subcellular localization of Nuk indicates that this receptor is concentrated at sites of cell-cell contact, often involving migrating neuronal cells or their extensions. Most notably, high levels of Nuk protein are found within initial axon outgrowths and associated nerve fibers. The axonal localization of Nuk is transient and is not detected after migrations have ceased, suggesting a role for this tyrosine kinase during the early pathfinding and/or fasciculation stages of axonogenesis. The subcellular localization of Nuk, as well as the presence of fibronectin type III and immunoglobulin-like adhesive domains on the extracellular region, suggest this receptor tyrosine kinase may function to regulate specific cell-cell interactions during early development of the murine nervous system.

Amino Acid Sequence↗

PubMed1992

Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated receptor-target interactions.

Efficient binding of active phosphatidylinositol (PI) 3'-kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF-1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3'-kinase, the isolated CSF-1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF-1R KI bound PI 3'-kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3'-kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF-1R KI. Binding of the CSF-1R KI to PI 3'-kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF-1R with PI 3'-kinase in mammalian cells. Complex formation between the CSF-1R and PI 3'-kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha.

Amino Acids↗

PubMed1992

A mammalian dual specificity protein kinase, Nek1, is related to the NIMA cell cycle regulator and highly expressed in meiotic germ cells.

Screening of mouse cDNA expression libraries with antibodies to phosphotyrosine resulted in repeated isolation of cDNAs that encode a novel mammalian protein kinase of 774 amino acids, termed Nek1. Nek1 contains an N-terminal protein kinase domain which is most similar (42% identity) to the catalytic domain of NIMA, a protein kinase which controls initiation of mitosis in Aspergillus nidulans. In addition, both Nek1 and NIMA have a long, basic C-terminal extension, and are therefore similar in overall structure. Despite its identification with anti-phosphotyrosine antibodies, Nek1 contains sequence motifs characteristic of protein serine/threonine kinases. The Nek1 kinase domain, when expressed in bacteria, phosphorylated exogenous substrates primarily on serine/threonine, but also on tyrosine, indicating that Nek1 is a dual specificity kinase with the capacity to phosphorylate all three hydroxyamino acids. Like NIMA, Nek1 preferentially phosphorylated beta-casein in vitro. In situ RNA analysis of nek1 expression in mouse gonads revealed a high level of expression in both male and female germ cells, with a distribution consistent with a role in meiosis. These results suggest that Nek1 is a mammalian relative of the fungal NIMA cell cycle regulator.

Amino Acid Sequence↗

PubMed1992

Multiple cDNAs encoding the esk kinase predict transmembrane and intracellular enzyme isoforms.

A novel protein kinase, the Esk kinase, has been isolated from an embryonal carcinoma (EC) cell line by using an expression cloning strategy. Sequence analysis of two independent cDNA clones (2.97 and 2.85 kb) suggested the presence of two Esk isoforms in EC cells. The esk-1 cDNA sequence predicted an 857-amino-acid protein kinase with a putative membrane-spanning domain, while the esk-2 cDNA predicted an 831-amino-acid kinase which lacked this domain. In adult mouse cells, esk mRNA levels were highest in tissues possessing a high proliferation rate or a sizeable stem cell compartment, suggesting that the Esk kinase may play some role in the control of cell proliferation or differentiation. As anticipated from the screening procedure, bacterial expression of the Esk kinase reacted with antiphosphotyrosine antibodies on immunoblots. Furthermore, in in vitro kinase assays, the Esk kinase was shown to phosphorylate both itself and the exogenous substrate myelin basic protein on serine, threonine, and tyrosine residues, confirming that the Esk kinase is a novel member of the serine/threonine/tyrosine family of protein kinases.

Amino Acid Sequence↗

PubMed1991

A mammalian protein kinase with potential for serine/threonine and tyrosine phosphorylation is related to cell cycle regulators.

In a screen of mouse erythroleukemia cDNA expression libraries with anti-phosphotyrosine antibodies, designed to isolate tyrosine kinase coding sequences, we identified several cDNAs encoding proteins identical or very similar to known protein-tyrosine kinases. However, two frequently isolated cDNAs, clk and nek, encode proteins which are most closely related to protein kinases involved in regulating progression through the cell cycle, and contain motifs generally considered diagnostic of protein-serine/threonine kinases. The clk gene product contains a C-terminal cdc2-like kinase domain, most similar to the FUS3 catalytic domain. The Clk protein, expressed in bacteria, becomes efficiently phosphorylated in vitro on tyrosine as well as serine/threonine, and phosphorylates the exogenous substrate poly(glu, tyr) on tyrosine. Direct biochemical evidence indicates that both protein-tyrosine and protein-serine/threonine kinase activities are intrinsic to the Clk catalytic domain. These results suggest the existence of a novel class of protein kinases, with an unusual substrate specificity, which may be involved in cell cycle control.

Amino Acid Sequence↗

PubMed1991

Erythroleukemia induction by Friend murine leukemia virus: insertional activation of a new member of the ets gene family, Fli-1, closely linked to c-ets-1.

The retroviral integration site Fli-1 is rearranged in 75% of the erythroleukemia cell clones induced by Friend murine leukemia virus (F-MuLV), whereas Spi-1/PU.1, a member of the ets family of DNA-binding proteins, is rearranged in 95% of the erythroleukemias induced by Friend spleen focus-forming virus (SFFV). To determine the transcriptional domain defined by Fli-1, we have isolated a cDNA clone that is highly expressed only in erythroleukemia cell lines with Fli-1 rearrangements. The protein sequence of this cDNA is very similar to Erg2, another member of the ets gene family. The hydrophilic carboxy-terminal end of the Fli-1 cDNA shares significant sequence similarity to the DNA-binding ETS domain found in all members of the ets family. PFGE analysis localized Fli-1 within 240 kb of the ets-1 proto-oncogene on mouse chromosome 9 and human chromosome 11q23, suggesting that ets-1 and Fli-1 arose from a common ancestral gene by gene duplication. The involvement of the murine Fli-1, Spi-1, and avian v-ets genes in erythroleukemia induction suggests that activation of ets gene family members plays an important role in the progression of these multistage malignancies.

Amino Acid Sequence↗

PubMed1991

Characterization of elk, a brain-specific receptor tyrosine kinase.

The elk gene encodes a novel receptorlike protein-tyrosine kinase, which belongs to the eph subfamily. We have previously identified a partial cDNA encompassing the elk catalytic domain (K. Letwin, S.-P. Yee, and T. Pawson, Oncogene 3:621-678, 1988). Using this cDNA as a probe, we have isolated cDNAs spanning the entire rat elk coding sequence. The predicted Elk protein contains all the hallmarks of a receptor tyrosine kinase, including an N-terminal signal sequence, a cysteine-rich extracellular domain, a membrane-spanning segment, a cytoplasmic tyrosine kinase domain, and a C-terminal tail. In both amino acid sequence and overall structure, Elk is most similar to the Eph and Eck protein-tyrosine kinases, suggesting that the eph, elk, and eck genes encode members of a new subfamily of receptorlike tyrosine kinases. Among rat tissues, elk expression appears restricted to brain and testes, with the brain having higher levels of both elk RNA and protein. Elk protein immunoprecipitated from a rat brain lysate becomes phosphorylated on tyrosine in an in vitro kinase reaction, consistent with the prediction that the mammalian elk gene encodes a tyrosine kinase capable of autophosphorylation. The characteristics of the Elk tyrosine kinase suggest that it may be involved in cell-cell interactions in the nervous system.

Amino Acid Sequence↗

PubMed1989

The FER gene is evolutionarily conserved and encodes a widely expressed member of the FPS/FES protein-tyrosine kinase family.

We have recently isolated human and rat cDNAs (designated FER and flk, respectively) which encode nonreceptor protein-tyrosine kinases which are very similar to one another and related in sequence and domain structure to the c-fps/fes gene product. We show that FER and flk are human and rat counterparts of an evolutionarily conserved gene, hereafter termed FER regardless of species. The human and rat FER genes encode a widely expressed 94-kilodalton protein-tyrosine kinase which is antigenically related to the fps/fes protein-tyrosine kinase. The structural and antigenic similarities between the FER and fps/fes proteins suggest that they are members of a new family of nonreceptor protein-tyrosine kinases.

Animals↗

PubMed1988

Novel protein-tyrosine kinase cDNAs related to fps/fes and eph cloned using anti-phosphotyrosine antibody.

A rat brain lambda gt11 cDNA expression library was screened with anti-phosphotyrosine antibody to identify recombinant clones that encode enzymatically active protein-tyrosine kinases. The inserts of two bacteriophage that gave positive signals were sequenced. Both translation products possess sequence motifs characteristic of protein-tyrosine kinases. However, each polypeptide is distinct from previously described members of the tyrosine kinase family. The predicted product of the lambda B1 clone contains a catalytic domain and C-terminal tail most closely related to the eph gene product, a presumed transmembrane receptor-like protein-tyrosine kinase. The clone lambda B2 encodes a partial SH2 domain and a kinase domain similar in organization and sequence to the fps/fes cytoplasmic protein-tyrosine kinase. These new protein-tyrosine kinases (elk and flk) are apparently members of subfamilies for which eph and fps/fes are prototypes. elk is predominantly expressed in brain, while flk RNA is widely distributed and most abundant in testes. The preferential isolation of cDNAs for previously uncharacterized protein-tyrosine kinases in a screen based on catalytic activity suggests that additional members of the protein-tyrosine kinase family remain to be identified.

Amino Acid Sequence↗