The Novel Inhibitor PLX3397 Effectively Inhibits FLT3-Mutant AML,

Abstract Abstract 3632 Mutations of FLT3 (FMS-like tyrosine kinase 3) are frequently present in acute myeloid leukemias (AML) and associated with poor prognosis. Such mutations alter the conformation and constitutively activate the FLT3 tyrosine kinase. PLX3397 is a novel, orally active, selective small molecule inhibitor of the FLT3-mutant as well as FMS and KIT kinases. The high selectivity of PLX3397 has been demonstrated in biochemical assays against a panel of over 200 recombinant kinases. FLT3, FMS and KIT are significantly inhibited at low nanomolar concentrations. The majority of kinases screened were not inhibited by PLX3397 (IC50=>10 μM) and the only other kinase with significant sub-micromolar inhibition was KDR. PLX3397 exhibits favorable pharmaceutical properties and demonstrated minimal off-target activity when tested in a broad array of 71 targets in 8 families (Neurotransmitter-related, Steroids, Ion Channels, Nitric Oxide, Prostaglandins, Growth Factors, Brain/Gut Peptides, and Enzymes). In cellular assays PLX3397 effectively inhibited ligand-stimulated autophosphorylation of the endogenous receptor tyrosine kinases FMS (IC50=20 nM) and KIT (IC50=120 nM), but not ligand-stimulated wild-type FLT3 (IC50=1.7 μM). In SEMK2 cells, which over-express wild type FLT3, PLX3397 inhibited FLT3 autophosphorylation with an IC50 of 240 nM. The phosphorylation of the activated FLT3-ITD protein in MV-4-11 and MOLM-14 cells harboring mutations (FLT3-ITD) of the juxtamembrane region of FLT3 was efficiently inhibited (IC50 26 nM and 30 nM respectively), indicating a possible application in this major subset of FLT3 mutated AML. In parallel with inhibition of FLT3-mutant autophosphorylation, the phosphorylation of known downstream effectors (STAT5, AKT, MEK, and ERK) is also inhibited. However, no inhibition was observed for KDR phosphorylation. PLX3397 potently inhibited of mutant-FLT3 driven proliferation in both MV-4-11 and MOLM-14 AML cells in culture (IC50=100–200 nM), but was less effective against HL60 cells expressing wild-type FLT3. MV-4-11 cells grown as subcutaneous xenograft tumors in mice were also highly sensitive to PLX3397 dosed orally at 10 or 30 mg/kg qd, with tumor regression observed at the higher dose. Primary samples collected from AML patients at relapse were tested in culture. While no significant effects were seen in FLT3 wild type samples below 1 μM, a clear dose response to PLX3397 was observed in samples with FLT3-ITD mutations. Because the estimated protein binding of PLX3397 in plasma is >99%, we also tested the drug against the MV-4-11 cell line in plasma and found the IC50 for inhibition of autophosphorylation to be 2.8 μM. In humans trough levels of PLX3397 (10 μM) capable of inhibiting FLT3-mutants can be safely achieved. A Phase I/II study is planned to evaluate the safety and explore the efficacy of PLX3397 in patients with FLT3-mutant- AML. Disclosures: Burton: Plexxikon Inc.: Employment. Wong:Plexxikon Inc.: Employment. Zhang:Plexxikon Inc.: Employment. West:Plexxikon Inc.: Employment. Bollag:Plexxikon Inc.: Employment. Habets:Plexxikon Inc.: Employment. Nguyen:Plexxikon Inc.: Employment. Levis:Plexxikon Inc.: Honoraria.

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Activation of pp60(src) is critical for stretch-induced orienting response in fibroblasts

Journal of Cell Science ◽

10.1242/jcs.112.9.1365 ◽

1999 ◽

Vol 112 (9) ◽

pp. 1365-1373 ◽

Cited By ~ 5

Author(s):

X. Sai ◽

K. Naruse ◽

M. Sokabe

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Time Course ◽

Kinase Inhibitor ◽

Orienting Response ◽

Cyclic Stretch ◽

Wild Type ◽

Herbimycin A ◽

Molecular Masses

When subjected to uni-axial cyclic stretch (120% in length, 1 Hz), fibroblasts (3Y1) aligned perpendicular to the stretch axis in a couple of hours. Concomitantly with this orienting response, protein tyrosine phosphorylation of cellular proteins (molecular masses of approximately 70 kDa and 120–130 kDa) increased and peaked at 30 minutes. Immuno-precipitation experiments revealed that paxillin, pp125(FAK), and pp130(CAS) were included in the 70 kDa, and 120–130 kDa bands, respectively. Treatment of the cells with herbimycin A, a tyrosine kinase inhibitor, suppressed the stretch induced tyrosine phosphorylation and the orienting response suggesting that certain tyrosine kinases are activated by stretch. We focused on pp60(src), the most abundant tyrosine kinase in fibroblasts. The kinase activity of pp60(src) increased and peaked at 20 minutes after the onset of cyclic stretch. Treatment of the cells with an anti-sense S-oligodeoxynucleotide (S-ODN) against pp60(src), but not the sense S-ODN, inhibited the stretch induced tyrosine phosphorylation and the orienting response. To further confirm the involvement of pp60(src), we performed the same sets of experiments using c-src-transformed 3Y1 (c-src-3Y1) fibroblasts. Cyclic stretch induced a similar orienting response in c-src-3Y1 to that in wild-type 3Y1, but with a significantly faster rate. The time course of the stretch-induced tyrosine phosphorylation was also much faster in c-src-3Y1 than in 3Y1 fibroblasts. These results strongly suggest that cyclic stretch induces the activation of pp60(src) and that pp60(src) is indispensable for the tyrosine phosphorylation of pp130(CAS), pp125(FAK) and paxillin followed by the orienting response in 3Y1 fibroblasts.

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Induction of autophagy across multiple tumor types through irreversible HER tyrosine kinase blockade.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.e13517 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. e13517-e13517

Author(s):

William Rayford Gwin ◽

Leihua Liu ◽

Sumin Zhao ◽

Wenle Xia ◽

Neil Spector

Keyword(s):

Prostate Cancer ◽

Cell Cycle ◽

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Cellular Proliferation ◽

Kinase Inhibitor ◽

Significant Inhibition ◽

Wild Type ◽

Multiple Tumor ◽

Mtorc1 Signaling

e13517 Background: Human epidermal growth factor receptor (HER) receptor tyrosine kinases play a key role in solid tumor oncogenesis. Despite broad expression of HER receptors in solid tumors, HER targeted therapies have not shown significant improvement in survival, calling into question the value of wild-type HER receptors as therapeutic targets. Here we found that an irreversible pan-HER tyrosine kinase inhibitor (TKI), neratinib, but not similar HER TKIs, induced morphologic changes in ovarian, TNBC, and prostate cancer cell lines consistent with induction of autophagy. Methods: SKOV3 (ovarian), OVCAR8 (ovarian), HBL-100 (TNBC), and LAPC4 (prostate) cancer cells were treated with lapatinib, gefitinib, CI-1033, afatinib, and neratinib (0.5mM-2.5mM). The activation state of HER2, EGFR, HER3, Akt, Erk, p70S6, 4EBP1, and Ulk1 was determined by Western blot analysis (WB) at various time points of neratinib treatment. LC3 was analyzed by immunofluorescence (IF) microscopy and WB. Analysis of proliferation, apoptosis, and cell cycle were performed using WST-1, annexin V, and PI staining, respectively. Results: Neratinib, but not similar HER TKIs, induced marked cytoplasmic vacuolization in tumors. The conversion of LC3-I to LC3-II in neratinib-treated cells was consistent with induction of autophagy. Moreover, PI3K/Akt, MAPK/Erk1/2 and mTORC1 signaling cascades were inhibited in neratinib-treated cells, and were associated with the inhibition of phospho-Ulk1, a key step in autophagy initiation. Treatment with neratinib alone resulted in G1 cell cycle arrest. Importantly, the combination of neratinib and chloroquine, an autophagy inhibitor, induced a statistically significant inhibition of cellular proliferation (p <0.01) and increased apoptosis compared to treatment with either drug alone. Conclusions: Our data suggest that more effective inhibition of wild-type HER receptors, can lead to mTORC1 inhibition, which in turn triggers autophagy. Here, autophagy appears to protect cells rather than inducing apoptosis. Consequently, targeting both HER receptors and autophagy represents an attractive therapeutic strategy to treat tumors expressing wild-type HER receptors.

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Direct binding to and tyrosine phosphorylation of the alpha subunit of the type I interferon receptor by p135tyk2 tyrosine kinase

Molecular and Cellular Biology ◽

10.1128/mcb.14.12.8133-8142.1994 ◽

1994 ◽

Vol 14 (12) ◽

pp. 8133-8142 ◽

Cited By ~ 1

Author(s):

O Colamonici ◽

H Yan ◽

P Domanski ◽

R Handa ◽

D Smalley ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Kinase Assay ◽

Alpha Subunit ◽

Recombinant Baculoviruses ◽

Type I ◽

Receptor Subunit ◽

Juxtamembrane Region

Binding of type I interferons (IFNs) to their receptors induces rapid tyrosine phosphorylation of multiple proteins, including the alpha and beta subunits of the receptor, the polypeptides that form the transcriptional activator ISGF3 alpha (Stat113, Stat84, and Stat91), and the p135tyk2 and Jak-1 tyrosine kinases. In this report, we demonstrate that the alpha subunit of the type I IFN receptor (IFN-R) corresponds to the product of a previously cloned receptor subunit cDNA and, further, that the p135tyk2 tyrosine kinase directly binds and tyrosine phosphorylates this receptor subunit. Glutathione S-transferase (GST) fusion proteins encoding the different regions of the cytoplasmic domain of the alpha subunit can bind the p135tyk2 contained in human cell lysates. The association between the alpha subunit and Tyk2 was demonstrated by immunoblotting with anti-Tyk2 and antiphosphotyrosine antibodies and by using an in vitro kinase assay. Analogous experiments were then performed with recombinant baculoviruses encoding constitutively active Jak family tyrosine kinases. In this case, p135tyk2, but not Jak-1 or Jak-2 protein, binds to the GST-IFN-R proteins, suggesting that the interaction between these two proteins is both direct and specific. We also demonstrate that Tyk2, from extracts of either IFN alpha-treated human cells or insect cells infected with the recombinant baculoviruses, can catalyze in vitro phosphorylation of GST-IFN-R protein in a specific manner. Deletion mutants of the GST-IFN-R protein were used to localize both the binding and tyrosine phosphorylation site(s) to a 46-amino-acid juxtamembrane region of the alpha subunit, which shows sequence homology to functionally similar regions of other cytokine receptor proteins. These data support the hypothesis that the Tyk2 protein functions as part of a receptor complex to initiate intracellular signaling in response to type I IFNs.

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Identification of Novel Phosphorylation Sites and Kinetics in Flt3 Receptor Wt, ITD and D835Y

Blood ◽

10.1182/blood.v112.11.5395.5395 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5395-5395

Author(s):

Elena Razumovskaya ◽

Kristina Masson ◽

Rasheed Khan ◽

Susanne Bengtsson ◽

Lars Ronnstrand

Keyword(s):

Tyrosine Kinase ◽

Gene Mutations ◽

Kinase Domain ◽

Phosphorylation Sites ◽

Dependent Manner ◽

Wild Type ◽

Over Expression ◽

Juxtamembrane Region ◽

Biological Responses

Abstract FMS-like tyrosine kinase-3 (Flt3) is a receptor tyrosine kinase, which is normally expressed in hematopoietic progenitor cells. It has been implicated as a major cause of transformation in acute myeloid leukemia (AML). There are two types of Flt3 gene mutations have been identified in AML: duplication of amino acids in the juxtamembrane region- Internal Tandem Duplication (ITD) and an activation loop point-mutation of D835 in kinase domain. These mutations cause constitutive activation or over expression of Flt3 receptor and therefore lead to alteration in signal transduction. These alterations occur in approximately 30% of AML patients. High occurrence of these mutations in the Flt3 receptor in AML patients makes it one of the most interesting therapeutic targets. In this study we have identified three novel in vivo phosphorylation sites of Flt3 receptor and further compared the activity of phosphorylation sites of Flt3 wild type, Flt3 ITD and D835Y mutations by using homemade phospho-specific antibodies directed against specific tyrosines. For this study murine hematopoietic Ba/F3 cells were stably transfected with wild-type Flt3, ITD and D835Y mutations. We have confirmed that the activation of the wild type Flt3 receptor is ligand dependent and response in a time dependent manner, but Flt3-ITD and D835Y are constitutive active and ligand independent. Phosphorylated tyrosines 589, 591, 599, 726, 768, 793, 842, and 955 of Flt3 receptor were investigated and shown to be differentially activated in wild-type versus the mutated receptor. Using this data we can further study the mechanisms of signaling pathways of the Flt3 receptor that are involved in many biological responses and understand the mechanism by which Flt3 ITD and D835Y functions in pathological conditions.

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A tyrosine kinase protein interaction map reveals targetable EGFR network oncogenesis in lung cancer

10.1101/2020.07.02.185173 ◽

2020 ◽

Author(s):

Swati Kaushik ◽

Franziska Haderk ◽

Xin Zhao ◽

Hsien-Ming Hu ◽

Khyati N. Shah ◽

...

Keyword(s):

Lung Cancer ◽

Tyrosine Kinase ◽

Protein Interaction ◽

Tyrosine Kinases ◽

Mapk Pathway ◽

Affinity Purification ◽

List Type ◽

Wild Type ◽

Interaction Map

SUMMARYSignaling networks balance the activities of many physically interacting proteins and perturbations to this network influence downstream signaling, potentially leading to oncogenic states. Using affinity purification-mass spectrometry we defined this network for all 90 human tyrosine kinases revealing 1,463 mostly novel interactions between these key cancer proteins and diverse molecular complexes. Modulation of interactor levels altered growth phenotypes associated with corresponding tyrosine kinase partners suggesting that tumors may alter the stoichiometries of interactors to maximize oncogenic signaling. We show that the levels of EGFR interactors delineates this form of network oncogenesis in 19% of EGFR wild-type lung cancer patients which were mostly otherwise oncogene negative, predicting sensitivity to EGFR inhibitors in vitro and in vivo. EGFR network oncogenesis occurs through mechanistically distinct network alleles often in cooperation with weak oncogenes in the MAPK pathway. Network oncogenesis may be a common and targetable convergent mechanism of oncogenic pathway activation in cancer.HIGHLIGHTSA human tyrosine kinome protein interaction map reveals novel physical and functional associations.Dependence on oncogenic tyrosine kinases is modulated through perturbation of their interactors.EGFR network oncogenesis in up to 19% of EGFR wild-type lung cancers is targetable.EGFR network oncogenesis cooperates with weak oncogenes in the MAPK pathway.

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PDGF alpha-receptor mediated cellular responses are not dependent on Src family kinases in endothelial cells

Journal of Cell Science ◽

10.1242/jcs.111.5.607 ◽

1998 ◽

Vol 111 (5) ◽

pp. 607-614

Author(s):

R. Hooshmand-Rad ◽

K. Yokote ◽

C.H. Heldin ◽

L. Claesson-Welsh

Keyword(s):

Endothelial Cells ◽

Tyrosine Kinases ◽

Src Family Kinases ◽

Wild Type ◽

Actin Reorganization ◽

Aortic Endothelial Cells ◽

Mutant Receptor ◽

Juxtamembrane Region ◽

Src Family Tyrosine Kinases ◽

Type Receptor

Two novel autophosphorylation sites in the juxtamembrane region of the PDGF alpha-receptor, Tyr-572 and Tyr-574, were identified. A Y572/574F mutant PDGF (alpha)-receptor was generated and stably expressed in porcine aortic endothelial cells. In contrast to the wild-type receptor, the mutant receptor was unable to associate with or activate Src family tyrosine kinases. Tyrosine phosphorylated synthetic peptides representing the juxtamembrane sequence of the receptor dose-dependently inhibited the binding of Src family tyrosine kinases to the autophosphorylated PDGF alpha-receptor. The mutant receptor showed similar PDGF-induced kinase activity and ability to mediate mitogenicity, actin reorganization and chemotaxis as the wild-type receptor. Thus activation of Src family kinases by the PDGF alpha-receptor is not essential for PDGF-induced mitogenicity or actin reorganization.

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Degrasyn-Induced Trafficking of BCR-ABL as a Novel Mechanism of Kinase Inactivation.

Blood ◽

10.1182/blood.v110.11.1003.1003 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1003-1003

Author(s):

Nicholas Donato ◽

Vaibhav Kapuria ◽

Hanshi Sun ◽

David Maxwell ◽

Geoffrey Bartholomeusz ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Point Mutations ◽

Binding Pocket ◽

Atp Binding ◽

Wild Type ◽

Post Translational Modification ◽

Oncogenic Signaling ◽

Imatinib Resistance ◽

Unique Mechanism

Abstract Inhibitors that inactivate specific tyrosine kinases have proven to be a very effective form of therapy of many leukemias and hematopoetic disorders. Most inhibitors function by competing for the ATP-binding pocket or by preventing association with protein substrates. However, clinical and molecular studies have shown that small changes in the structure of the target kinase (point mutations, post-translational modification) affect inhibitor binding affinities, resulting in resistance to this class of inhibitor. Therefore, development of agents that reduce the activity of leukemogenic kinases through alternate mechanisms are of great interest. We previously described a novel class of compounds, termed degrasyns, which reduced BCR-ABL (and Jak2) kinase activity through a unique mechanism. Degrasyn treatment of CML or BaF3 cells expressing wild-type or mutant (T315I) BCR-ABL resulted in a reduction of cytoplasmic BCR-ABL protein levels and loss of downstream signaling without a direct effect on BCR-ABL enzymatic activity. Biochemical studies demonstrated that degrasyn induced a rapid translocation of BCR-ABL from the cytosol to the cytoskeletal fraction (complete within 60 min) and this event correlated with loss of BCR-ABL signaling and initiation of apoptosis. A GFP-fusion protein composed of wild-type or T315I mutant BCR-ABL demonstrated that degrasyn induced high density translocation to the cytoskeletal fraction, as determined by direct fluorescence imaging. Translocation was specific for BCR-ABL and not other kinases (except Jak2) or signaling proteins. Translocation correlated with degrasyn-mediated tyrosine phosphorylation of a subset of specific proteins, including the tyrosine kinase Lyn, in the insoluble cellular fraction. CML cells expressing high levels of Lyn were more sensitive to degrasyn-mediated apoptosis suggesting that Lyn plays a role in degrasyn activity. Together these results suggest that degrasyn inactivates BCR-ABL by inducing its translocation to a cellular compartment that prevents its participation in oncogenic signaling. Degrasyn-induced BCR-ABL translocation was not effected by mutations that block dasatinib and imatinib activity and shows greater activity against some forms of imatinib resistance (Lyn overexpression). Animal studies demonstrated that degrasyn has anti-leukemic activity and functions through a unique mechanism of action. Development of inhibitors with this mode of action may be of significance for CML patients that fail therapy with ATP-binding pocket-directed tyrosine kinase inhibitors.

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Small-Molecule Inhibitor Screen Rapidly Identifies Key Signaling Pathways in Leukemogenesis

Blood ◽

10.1182/blood.v112.11.2519.2519 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2519-2519

Author(s):

Jeffrey W Tyner ◽

Heidi Erickson ◽

Stephen Oh ◽

Jason R. Gotlib ◽

Michael W.N. Deininger ◽

...

Keyword(s):

Tyrosine Kinase ◽

Cell Viability ◽

Small Molecule ◽

Tyrosine Kinases ◽

Small Molecule Inhibitor ◽

Wild Type Allele ◽

Wild Type ◽

Kinase Activation ◽

Type Allele ◽

Molecule Inhibitor

Abstract Aberrantly activated tyrosine kinases and their associated signaling pathways are critical to leukemogenesis and primary acute myeloid leukemia (AML) cell viability. While aberrant kinase activation has been confirmed in a significant percentage of AML, constitutive phosphorylation of STAT5, a marker of tyrosine kinase activation, is present in the majority of AML samples indicating that as yet unidentified tyrosine kinases can be aberrantly activated and contribute to leukemogenesis. Efforts to identify activating tyrosine kinase mutations using high-throughput sequencing have identified low frequency mutations of uncertain functional significance. Because these studies failed to detect additional high-frequency kinase mutations, the identity and mechanism of tyrosine kinase activation may be unique in many AMLs. Methods: To rapidly identify activated kinase pathways in individual, primary AML samples, we have developed a small-molecule inhibitor array which includes 90 small-molecule, cell-permeable inhibitor compounds including a core of 36 tyrosine kinase inhibitors that covers the majority of the tyrosine kinome. Many of the inhibitors are available for clinical use or are in clinical development. In this assay, inhibitors were placed in 96-well plates at four serial dilutions to allow IC50 calculations. Three days after adding primary AML cells to each well, we performed an MTS cell viability assay to evaluate the effect of each inhibitor on cell viability. Because most inhibitors affect multiple kinases, we compared target specificities of compounds that decrease primary AML cell viability with those that have no effect to identify potential targets. Results: In preliminary proof-of-principal experiments, we tested leukemic cells from five AML patients, including three that were positive for FLT3 internal tandem duplication (ITD), a genetic lesion that is thought to confer a proliferative advantage in approximately 30% of AML patients. Only one sample showed a clear response to small-molecule inhibitors known to target FLT3. The IC50s for the known FLT3 inhibitors MLN518, AST487, CHIR258, Sunitinib, and SU14813 were 10 to 100 fold lower in the leukemic cells from this patient than for the mean and median values for bone marrow samples from normal marrow samples. Interestingly, neither of the remaining FLT3-ITD positive leukemias nor the FLT3 negative leukemias demonstrated decreased viability in the presence of small-molecule inhibitors that target FLT3. A PCR based screen to identify FLT3-ITD alleles showed a near absence of the wild-type FLT3 allele in this sample, while the wild-type allele was present at equal or greater intensity as the ITD allele in the remaining FLT3-ITD positive AML samples. FLT3-ITD positive leukemias with loss of the FLT3 wild-type allele have been shown to have a poorer prognosis than those retaining a wild-type allele indicating aberrantly activated FLT3 may play a crucial role in leukemic cell viability in this setting and are consistent with a previous report showing that efficacy of the FLT3 inhibitor CEP-701 was greatest in pediatric AML with high FLT3-ITD mutant-to-wild-type allelic ratios. Though the remaining AML samples did not show a pattern of inhibitor sensitivity consistent with FLT3 activation, these and many of approximately 15 additional analyzed AML samples showed unique sensitivity patterns implicating other specific kinase targets or kinase families for further investigation while simultaneously providing therapeutic options. Conclusions: These preliminary data demonstrate that the small-molecule inhibitor functional assays can rapidly identify disease causing genes, provide insights into their mechanism of action, and suggest therapeutic options. The distinct patterns of tyrosine kinase sensitivity in these samples support the hypothesis that tyrosine kinases and related pathways contributing to leukemogenesis in each patient may be different and that targeted therapy will be most effective when administered on an individualized basis.

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A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4396-4408.1986 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4396-4408

Author(s):

I Sadowski ◽

J C Stone ◽

T Pawson

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Protein Tyrosine Kinases ◽

Cytoplasmic Protein ◽

Avian Sarcoma Virus ◽

Wild Type ◽

Protein Tyrosine ◽

Terminal Domain ◽

Sarcoma Virus ◽

Avian Sarcoma

Proteins encoded by oncogenes such as v-fps/fes, v-src, v-yes, v-abl, and v-fgr are cytoplasmic protein tyrosine kinases which, unlike transmembrane receptors, are localized to the inside of the cell. These proteins possess two contiguous regions of sequence identity: a C-terminal catalytic domain of 260 residues with homology to other tyrosine-specific and serine-threonine-specific protein kinases, and a unique domain of approximately 100 residues which is located N terminal to the kinase region and is absent from kinases that span the plasma membrane. In-frame linker insertion mutations in Fujinami avian sarcoma virus which introduced dipeptide insertions into the most stringently conserved segment of this N-terminal domain in P130gag-fps impaired the ability of Fujinami avian sarcoma virus to transform rat-2 cells. The P130gag-fps proteins encoded by these transformation-defective mutants were deficient in protein-tyrosine kinase activity in rat cells. However v-fps polypeptides derived from the mutant Fujinami avian sarcoma virus genomes and expressed in Escherichia coli as trpE-v-fps fusion proteins displayed essentially wild-type enzymatic activity, even though they contained the mutated sites. Deletion of the N-terminal domain from wild-type and mutant v-fps bacterial proteins had little effect on autophosphorylating activity. The conserved N-terminal domain of P130gag-fps is therefore not required for catalytic activity, but can profoundly influence the adjacent kinase region. The presence of this noncatalytic domain in all known cytoplasmic tyrosine kinases of higher and lower eucaryotes argues for an important biological function. The relative inactivity of the mutant proteins in rat-2 cells compared with bacteria suggests that the noncatalytic domain may direct specific interactions of the enzymatic region with cellular components that regulate or mediate tyrosine kinase function.

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