Exhibit Tyrosine Kinase Activity Research Articles

MTORC2 promotes type I insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mTOR

Mammalian target of rapamycin (mTOR) is a core component of raptor-mTOR (mTORC1) and rictor-mTOR (mTORC2) complexes that control diverse cellular processes. Both mTORC1 and mTORC2 regulate several elements downstream of type I insulin-like growth factor receptor (IGF-IR) and insulin receptor (InsR). However, it is unknown whether and how mTOR regulates IGF-IR and InsR themselves. Here we show that mTOR possesses unexpected tyrosine kinase activity and activates IGF-IR/InsR. Rapamycin induces the tyrosine phosphorylation and activation of IGF-IR/InsR, which is largely dependent on rictor and mTOR. Moreover, mTORC2 promotes ligand-induced activation of IGF-IR/InsR. IGF- and insulin-induced IGF-IR/InsR phosphorylation is significantly compromised in rictor-null cells. Insulin receptor substrate (IRS) directly interacts with SIN1 thereby recruiting mTORC2 to IGF-IR/InsR and promoting rapamycin- or ligand-induced phosphorylation of IGF-IR/InsR. mTOR exhibits tyrosine kinase activity towards the general tyrosine kinase substrate poly(Glu-Tyr) and IGF-IR/InsR. Both recombinant mTOR and immunoprecipitated mTORC2 phosphorylate IGF-IR and InsR on Tyr1131/1136 and Tyr1146/1151, respectively. These effects are independent of the intrinsic kinase activity of IGF-IR/InsR, as determined by assays on kinase-dead IGF-IR/InsR mutants. While both rictor and mTOR immunoprecitates from rictor(+/+) MCF-10A cells exhibit tyrosine kinase activity towards IGF-IR and InsR, mTOR immunoprecipitates from rictor(-/-) MCF-10A cells do not induce IGF-IR and InsR phosphorylation. Phosphorylation-deficient mutation of residue Tyr1131 in IGF-IR or Tyr1146 in InsR abrogates the activation of IGF-IR/InsR by mTOR. Finally, overexpression of rictor promotes IGF-induced cell proliferation. Our work identifies mTOR as a dual-specificity kinase and clarifies how mTORC2 promotes IGF-IR/InsR activation.

Protein kinase CK2 catalyzes tyrosine phosphorylation in mammalian cells

Protein kinase CK2 exhibits oncogenic activity in mice and is over-expressed in a number of tumors or leukemic cells. On the basis of its amino acid sequence and a wealth of experimental information, CK2 has traditionally been classified as a protein serine/threonine kinase. In contrast to this traditional view of CK2, recent evidence has shown that CK2 can also phosphorylate tyrosine residues under some circumstances in vitro and in yeast. In this study, we provide definitive evidence demonstrating that CK2 also exhibits tyrosine kinase activity in mammalian cells. Tyrosine phosphorylation of CK2 in cells and in CK2 immunoprecipitates is dependent on CK2 activity and is inhibited by the CK2 selective inhibitor 4,5,6,7-tetrabromobenzotriazole. Examination of phosphotyrosine profiles in cells reveals a number of proteins, including CK2 itself, which exhibit increased tyrosine phosphorylation when CK2 levels are increased. Peptide arrays to evaluate the specificity determinants for tyrosine phosphorylation by CK2 reveal that its specificity for tyrosine phosphorylation is distinct from its specificity for serine/threonine phosphorylation. Of particular note is the requirement for an aspartic acid immediately C-terminal to the phosphorylatable tyrosine residue. Collectively, these data provide conclusive evidence that CK2 catalyzes the phosphorylation of tyrosine residues in mammalian cells, a finding that adds a new level of complexity to the challenge of elucidating its cellular functions. Furthermore, these results raise the possibility that increased CK2 levels that frequently accompany transformation may contribute to the increased tyrosine phosphorylation that occurs in transformed cells.

FIP1L1/PDGFRA is a molecular marker of chronic eosinophilic leukaemia but not for systemic mastocytosis

Sir,During the past decade, a number of molecular markersspecific for haematological malignancies have been identi-fied. For chronic eosinophilic leukaemia (CEL) [1], onerecurrent molecular marker is the FIP1L1/PDGFRA fusiongene product that exhibits tyrosine kinase activity and rep-resents a target of imatinib unless the gene is specificallymutated [2,3]. Over the past few years, an attendant dis-cussion about the relationship of FIP1L1/PDGFRA to sys-temic mastocytosis (SM) has ensued [3]. In this letter, wewish to provide the following comments to clarify this issue.In SM, the frequency of associated haematological non-mast cell disorders (AHNMD) is relatively high [4–6]. UsingKIT D816V as a marker of SM, it has been noted that theAHNMD usually arises from the same clone [7,8]. Even ifKIT D816V is not detected in the AHNMD, both malig-nancies may be of monoclonal origin. Because of therapeuticimplications, the consensus has been to incorporate a specificcategory of SM in the World Health Organization (WHO)classification of mastocytosis, termed SM-AHNMD [9,10].Now, a number of reports have commented that patientswith CEL often present with an increase in bone marrowmast cells [2,3]. These mast cells usually are spindle-shapedand display aberrant expression of CD25 [3], features thatotherwise are found only with mast cells in SM [9]. In mostCEL patients, however, criteria for SM [9,10] are notfulfilled. This is because the mast cells are diffusely dis-tributed, do not form aggregates and do not display KITD816V. Nevertheless, in some CEL patients, mast cells doform multifocal clusters consistent with the diagnosis SM[3]. These patients are appropriately classified as SM-CEL.However, in these patients, the molecular defect, FIP1L1/PDGFRA, is not indicative of SM, but is indicative of theAHNMD, i.e. CEL. It is noteworthy that in these patients,disease symptoms are due to increased eosinophils, and donot overlap with symptoms of SM. Imatinib therefore is pre-scribed in these patients to bring the eosinophil numberunder control, but not to treat the SM-component of thedisease, which is indolent. Interestingly, most patients withSM-CEL do not have a detectable KIT mutation, which isof importance as KIT D816V confers resistance againstimatinib. Thus, in most patients with typical (KIT D816V-positive) SM, therapy with imatinib is not recommended.Regardless of the presence or absence of FIP1L1/PDGFRA,patients with SM may present with eosinophilia [4,6,9,10].Thus, in SM, eosinophilia must be considered as a ‘pre-diagnostic’ checkpoint (SM-eo) at which molecular markersneed to be determined, but is not a final diagnosis or deci-sion point at which treatment with imatinib or other drugscan be recommended. Rather, only a complete staging andgrading of symptoms and findings [9,10] and a consecutivesearch for targets will yield the information necessary fortreatment decisions and drug-selection [3,9,11]. This isimportant as patients with indolent SM usually have a normallife expectancy and do not require cytoreductive or targeteddrugs, even when presenting with eosinophilia.Therefore, in order to establish the correct diagnosis andselect the appropriate approach to management, we recom-mend the use of WHO criteria [9,10] in all patients withSM, including those with eosinophilia (SM-eo).

Cytokine Receptor-independent, Constitutively Active Variants of STAT5

STAT (signal transducers and activators of transcription) proteins are dual function proteins, which participate in cytokine-mediated signal transduction events at the cell surface and transcriptional regulation in the nucleus. We have exploited insights into the activation mechanism of STAT factors to derive constitutively active variants. Chimeric genes encoding fusion proteins of STAT5 and the kinase domain of JAK2 have been derived. The functional properties of the fusion proteins have been investigated in transiently transfected COS cells or in HeLa cells stably transfected with STAT5-JAK2 gene constructs regulated by a tetracycline-sensitive promoter. The STAT5-JAK2 proteins exhibit tyrosine kinase activity and are phosphorylated on tyrosine. The molecules are activated through an intramolecular or a cross-phosphorylation reaction and exhibit constitutive, STAT5-specific DNA binding activity. The transactivation potentials of three constitutively activated STAT5-JAK2 variants comprising different transactivation domains (TADs) derived from STAT5, STAT6, and VP16 were compared. The chimeric molecule containing the STAT5 TAD had no or only a very low, the molecule with the STAT6 TAD a medium, and the molecule with the VP16 TAD a very high transactivation potential. Transcription from STAT5-responsive gene promoter regions of the beta-casein, oncostatin M, and the cytokine-inducible Src homology 2 domain-containing protein genes was observed. These chimeric STAT molecules allow the study of the function of STAT5 independent of cytokine receptors and the activation of other signal transduction pathways.

Essential role of tyrosine residues 1131, 1135, and 1136 of the insulin-like growth factor-I (IGF-I) receptor in IGF-I action.

The insulin and insulin-like growth factor-I (IGF-I) receptors are related heterotetramers consisting of two extracellular ligand-binding alpha-subunits and two transmembrane beta-subunits whose cytoplasmic domains exhibit tyrosine kinase activity. Previous studies have shown that ATP binding by the cytoplasmic tyrosine kinase domains of these receptors is necessary to initiate the signal transduction pathway triggered by ligands or by ligand-mimetic antibodies, suggesting that receptor autophosphorylation is a necessary proximal step in this pathway. In the case of the insulin receptor, it has additionally been demonstrated that a cluster of three tyrosines in the kinase domain itself are the first to be phosphorylated, and that autophosphorylation of these particular residues is necessary for receptor activity. Using stably transfected NIH-3T3 cell lines, we now show that mutation of the analogous residues in the IGF-I receptor abolishes all short, intermediate, and long-term responses to IGF-I. These data suggest that the initial mechanisms of activation of the insulin and IGF-I receptors are very similar. Additionally, we have identified two parameters, induction of c-fos gene expression and ornithine decarboxylase enzyme activity, which are extremely sensitive to IGF-I stimulation and which will be particularly useful in evaluating the biological activity of other mutated versions of the IGF-I receptor.

Antibodies to Cytoplasmic Sequences of Cloned Liver Growth Hormone (GH) Receptors Recognize GH Receptors Associated with Tyrosine Kinase Activity*

GH stimulates tyrosyl phosphorylation of GH receptors in 3T3-F442A fibroblasts, and highly purified GH receptor preparations exhibit tyrosine kinase activity. Paradoxically, however, the GH receptor cloned from liver exhibits no sequence similarity to receptors with known signal transduction mechanisms, including those exhibiting ligand-activated tyrosine kinase activity. These observations raise the possibility that there are two kinds of receptors for GH: the first represented by the cloned liver GH receptor, and the second by a tyrosine kinase-containing GH receptor. To inquire into the possibility of two distinct GH receptors, we determined whether the cloned liver GH receptor shares structural similarities with the tyrosine kinase-associated GH receptor. When the cloned rabbit liver GH receptor is expressed in human kidney 293 cells, it migrates with a mol wt appropriate for the tyrosine kinase-associated GH receptor, despite the calculated mol wt of the cloned GH receptor being 60,000 smaller than that of the tyrosine kinase-associated GH receptor. The recognition of tyrosine kinase-associated GH receptor by antipeptide antibodies to three different epitopes on the cytoplasmic domain of the cloned liver GH receptor was also tested. Tyrosyl phosphorylated [125I]human GH-receptor complexes were prepared by immunoprecipitation with phosphotyrosyl-binding antibody; this subpopulation of GH-receptor complexes was recognized by all three antipeptide antibodies. The antibodies also recognized similarly isolated tyrosyl phosphorylated GH-receptor complexes, which had been further phosphorylated in solution on tyrosyl residues upon addition of [gamma 32P] ATP. Furthermore, highly purified GH receptors prepared by sequential immunoprecipitation using phosphotyrosyl-binding antibody and any one of the three antipeptide antibodies incorporated 32P into tyrosyl residues upon the addition of [gamma 32P] ATP. These results provide evidence that tyrosine kinase-associated GH receptors share sequence similarity in the cytoplasmic domain with the cloned liver GH receptor. The cloned GH receptor and the tyrosine kinase-associated GH receptor, therefore, are likely to be the same receptor or related receptor isoforms.

PROTEIN TYROSINE KINASE SUBSTRATES IN RAT INTESTINAL MICROVILLUS MEMBRANES (MVM)

Many growth factor receptors and proto-oncogenes regulate cellular proliferentiation and differentiation by an intrinsic tyrosine kinase which phosphorylates specific cellular substrates. These phosphotyrosyl proteins (p-tyr) are likely to be second messengers which transduce growth factor responses. To characterize these potential mediators of intestinal growth, we present the first identification of p-tyr in rat intestinal MVM, which we have previously shown to exhibit tyrosine kinase activity. (Gastroenterol 94:A460, 1988). MVM, purified from small intestine of fetal and adult rats by the calcium precipitation technique, or their corresponding intestinal homogenate, were incubated for 10 min at 20°C in vitro in Hepes/MnCl2ATP/Na orthovanadate. P-tyr were identified by either immunoprecipitation or Western blot using a highly specific anti-phosphotyrosine monoclonal or polyclonal antibody, respectively. A 68 kDa protein was the most abundant p-tyr identified in MVM (fetal > adult) using either technique. Immunoprecipitation demonstrated a 36 kDa p-tyr in fetal MVM and a 33 kDa p-tyr in adult MVM. A few other p-tyr were identified by Western blot in fetal MVM but only the 68 kDa was present in adult MVM. These p-tyr were significantly enriched in MVM compared to homogenates. To determine if these p-tyr are present in vivo and to study their developmental expression, Western blots were performed on MVM prepared from fetal, suckling and adult rat intestine in the presence of vanadate and NaF. The 68 kDa p-tyr was present in all ages studied and in MVM of adult jejunum and ileum. 80, 90 and 175 kDa p-tyr, previously identified in vitro, demonstrated variable expression during development. The epidermal growth factor receptor (EGFR), a known phosphotyrosyl protein, was immunoprecipitated from fetal MVM with an anti-EGFR antibody, incubated with 35P-ATP and identified as a single band of MW 175 kDa by autoradiography, suggesting that the 175 kDa p-tyr expressed in MVM is the EGFR. CONCLUSIONS: 1) MVM tyrosine kinase(s) phosphorylate p-tyr in vitro and in vivo. 2) A 68 kDa protein is the most adundant p-tyr present in fetal and adult MVM. 3) The 175 kDa p-tyr identified in MVM in vivo appears to be the EGFR which is autophosphorylated.

Insulin and insulin-like growth factor receptors in the nervous system

Insulin and the insulin-like growth factors (I and II) are homologous peptides essential to normal metabolism as well as growth. These peptide hormones are present in the brain, and, based on biosynthetic labeling studies as well as evidence for local gene expression, they are synthesized by nervous tissue as well as being taken up by the brain from the peripheral circulation. Furthermore, the presence of insulin and IGF receptors in the brain, on both neuronal and glial cells, also suggests a role for these peptides in the nervous system. Thus, these ligands affect brain electrical activity, either as neurotransmitters or as neuromodulators, altering the release and re-uptake of other neurotransmitters. The insulin and IGF-I and -II receptors found in the brain exhibit a lower molecular weight than corresponding receptors on peripheral tissues, primarily caused by alterations in glycosylation. Despite these alterations, both brain insulin and IGF-I receptors exhibit tyrosine kinase activity in cell-free systems, as do their peripheral counterparts. Brain insulin and IGF-I receptors are developmentally regulated, with the highest levels appearing in fetal or perinatal life. However, the altered glycosylation of brain receptors does not appear until late in fetal development. The receptors are widely distributed in the brain, but especially enriched in the circumventricular organs, choroid plexus, hypothalamus, cerebellum, and olfactory bulb. These studies on the insulin and IGF receptor in brain, add strong support to the suggestion that insulin and IGFs are important neuroactive substances, regulating growth, development, and metabolism in the brain.

Similar control mechanisms regulate the insulin and type I insulin-like growth factor receptor kinases. Affinity-purified insulin-like growth factor I receptor kinase is activated by tyrosine phosphorylation of its beta subunit.

Insulin-like growth factor I (IGF-I) receptors are partially purified from human placenta by sequential affinity chromatography with wheat germ agglutinin-agarose and agarose derivatized with an IGF-I analog. Adsorption specificity to this affinity matrix demonstrates that low coupling ratios of IGF-I analog to agarose yield preparations that are highly selective in purifying IGF-I receptor with minimal cross-contamination by the insulin receptor present in the same placental extracts. Incubation of the immobilized IGF-I receptor preparation with [gamma-32P]ATP results in a marked phosphorylation of the receptor beta subunits, which appear as a doublet of Mr = 93,000 and 95,000 upon electrophoresis on dodecyl sulfate-polyacrylamide gels. The 32P-labeled receptor beta subunit doublet contains predominantly phosphotyrosine and to a much lesser extent phosphoserine and phosphothreonine residues. The immobilized IGF-I receptor preparation exhibits tyrosine kinase activity toward exogenous histone. The characteristics of the IGF-I receptor-associated tyrosine kinase are remarkably similar to those of the insulin receptor kinase. Thus, prior phosphorylation of the immobilized IGF-I receptor preparation with increasing concentrations of unlabeled ATP followed by washing to remove the unreacted ATP results in a progressive activation of the receptor-associated histone kinase activity. A maximal (10-fold) activation is achieved between 0.25 and 1 mM ATP. The concentration of ATP required for half-maximal (30 microM) activation of the IGF-I receptor kinase is similar to that of the insulin receptor kinase. Like the insulin receptor kinase, the elevated kinase activity of the phosphorylated IGF-I receptor is reversed following dephosphorylation of the receptor beta subunit with alkaline phosphatase. Furthermore, the phosphorylation of the IGF-I receptor beta subunit doublet is enhanced by 7-8-fold when reductant is included in the reaction medium, as is observed for the insulin receptor kinase. Significantly, the dose responses of both receptor types to reductant are identical. Both of the 32P-labeled IGF-I receptor beta subunit bands are resolved into six matching phosphopeptide fractions when the corresponding tryptic hydrolysates are resolved by reverse phase high pressure liquid chromatography. Significantly, four out of the six phosphopeptide fractions derived from the trypsinized IGF-I receptor beta subunits are chromatographically identical to those from the tryptic hydrolysates of 32P-labeled insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin-like growth factor receptors.

There are two types of insulin-like growth factor (IGF) receptors. The type I receptor generally binds IGF-I more tightly than IGF-II and also interacts weakly with insulin. The type II receptor prefers IGF-II over IGF-I and does not recognize insulin. The type I receptor is made up of an alpha binding subunit (Mr 130 000) and a beta subunit (Mr 95 000) probably organized as a heterotetramer (alpha 2 beta 2). The type II receptor consists of a single binding unit (Mr 250 000). IGF stimulates phosphorylation of the beta subunit of the type I receptor in whole cells and solubilized receptor preparations. Tyrosine kinase activity is associated with the type I receptor, resulting in autophosphorylation of the beta subunit and phosphorylation of exogenous substrates. In contrast, phosphorylation of the type II receptor in whole cells is less IGF-dependent, solubilized receptor preparations are not phosphorylated, and purified type II receptors do not exhibit tyrosine kinase activity toward the artificial substrate poly(Glu, Tyr)4:1. There are many similarities between the type I IGF receptor and the insulin receptor; however, different ligand-binding properties, subtle differences in the size of alpha and beta subunits, and immunoreactivity toward anti-receptor antibodies allow us to distinguish between these two receptors. The presence of both IGF receptors as well as insulin receptors on most cells and cross-reactivity of ligands for binding to these receptors present difficulties in assigning a particular biological response to a specific receptor. The type I receptor is down-regulated by ligand while in several cell types the type II receptor is rapidly up-regulated by insulin; the mechanism of up-regulation appears to be a translocation of type II receptors to the cell surface. There are two classes of serum binding proteins for IGF, a Mr 150 000 species found in adult blood and a Mr 40 000 species, which predominates in foetal blood. Like the type II receptor, IGF binding proteins do not bind insulin. The binding site on the type II receptor can be distinguished from the binding protein sites by a hybrid molecule Ainsulin-BIGF-I, which recognizes the binding protein but not the type II receptor. Binding proteins produced by cells in culture may cause confusion in the interpretation of experiments that are designed to study the binding of radiolabelled IGF to cell surface receptors in monolayer culture.