Substitution of the autophosphorylation site Thr516 with a negatively charged residue confers constitutive activity to mouse 3-phosphoinositide-dependent protein kinase-1 in cells

3-Phosphoinositide-dependent protein kinase-1 (PDK-1)is a serine/threonine kinase that has been found to phosphorylate and activate several members of the AGC protein kinase family including protein kinase B (Akt), p70 S6 kinase, and protein kinase Czeta. However, the mechanism(s) by which PDK-1 is regulated remains unclear. Here we show that mouse PDK-1 (mPDK-1) undergoes autophosphorylation in vitro on both serine and threonine residues. In addition, we have identified Ser(399) and Thr(516) as the major mPDK-1 autophosphorylation sites in vitro. Furthermore, we have found that these two residues, as well as Ser(244) in the activation loop, are phosphorylated in cells and demonstrated that Ser(244) is a major in vivo phosphorylation site. Abolishment of phosphorylation at Ser(244), but not at Ser(399) or Thr(516), led to a significant decrease of mPDK-1 autophosphorylation and kinase activity in vitro, indicating that autophosphorylation at Ser(399) or Thr(516) is not essential for mPDK-1 autokinase activity. However, overexpression of mPDK-1(T516E), but not of mPDK-1(S244E) or mPDK-1(S399D), in Chinese hamster ovary and HEK293 cells was sufficient to induce Akt phosphorylation at Thr(308) to a level similar to that of insulin stimulation. Furthermore, this increase in phosphorylation was independent of the Pleckstrin homology domain of Akt. Taken together, our results suggest that mPDK-1 undergoes autophosphorylation at multiple sites and that this phosphorylation may be essential for PDK-1 to interact with and phosphorylate its downstream substrates in vivo.     

Nox4 NAD(P)H oxidase mediates Src-dependent tyrosine phosphorylation of PDK-1 in response to angiotensin II: role in mesangial cell hypertrophy and fibronectin expression

Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to hypertrophy and extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces an increase in PDK-1 (3-phosphoinositide-dependent protein kinase-1) kinase activity that required its phosphorylation on tyrosine 9 and 373/376. Introduction into the cells of PDK-1, mutated on these tyrosine residues or kinase-inactive, attenuates Ang II-induced hypertrophy and fibronectin accumulation. Ang II-mediated PDK-1 activation and tyrosine phosphorylation (total and on residues 9 and 373/376) are inhibited in cells transfected with small interfering RNA for Src, indicating that Src is upstream of PDK-1. In cells expressing oxidation-resistant Src mutant C487A, Ang II-induced hypertrophy and fibronectin expression are prevented, suggesting that the pathway is redox-sensitive. Ang II also up-regulates Nox4 protein, and siNox4 abrogates the Ang II-induced increase in intracellular reactive oxygen species (ROS) generation. Small interfering RNA for Nox4 also inhibits Ang II-induced activation of Src and PDK-1 tyrosine phosphorylation (total and on residues 9 and 373/376), demonstrating that Nox4 functions upstream of Src and PDK-1. Importantly, inhibition of Nox4, Src, or PDK-1 prevents the stimulatory effect of Ang II on fibronectin accumulation and cell hypertrophy. This work provides the first evidence that Nox4-derived ROS are responsible for Ang II-induced PDK-1 tyrosine phosphorylation and activation through stimulation of Src. Importantly, this pathway contributes to Ang II-induced MC hypertrophy and fibronectin accumulation. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal hypertrophy and fibrosis.     

The Protein-tyrosine-phosphatase SHP2 is phosphorylated on serine residues 576 and 591 by protein kinase C isoforms alpha, beta 1, beta 2, and eta.

To study whether protein kinase C (PKC) isoforms can interact with protein-tyrosine-phosphatases (PTPs) which are connected to the insulin signaling pathway, we co-overexpressed PKC isoforms together with insulin receptor, docking proteins, and the PTPs SHP1 and SHP2 in human embryonic kidney (HEK) 293 cells. After phorbol ester induced activation of PKC isoforms alpha, beta 1, beta 2, and eta, we could show a defined gel mobility shift of SHP2, indicating phosphorylation on serine/threonine residues. This phosphorylation was not dependent on insulin receptor or insulin receptor substrate-1 (IRS-1) overexpression and did not occur for the closely related phosphatase SHP1. Furthermore, PKC phosphorylation of SHP2 was completely blocked by the PKC inhibitor bisindolylmaleimide and was not detectable when SHP2 was co-overexpressed with kinase negative mutants of PKC beta 1 and -beta 2. The phosphorylation also occurred on endogenous SHP2 in Chinese hamster ovary (CHO) cells stably overexpressing PKC beta 2. Using point mutants of SHP2, we identified serine residues 576 and 591 as phosphorylation sites for PKC. However, no change of phosphatase activity by TPA treatment was detected in an in vitro assay. In summary, SHP2 is phosphorylated on serine residues 576 and 591 by PKC isoforms alpha, beta 1, beta 2, and eta.     

Phosphorylation of the 105-kDa heat shock proteins, HSP105alpha and HSP105beta, by casein kinase II

The 105-kDa heat shock protein alpha (HSP105alpha) and HSP105beta are mammalian heat shock proteins that belong to the HSP105/HSP110 family. Both HSP105alpha and HSP105beta consist of acidic and basic isoforms. Here we report that the acidic isoforms are serine phosphorylated HSP105alpha or HSP105beta. Furthermore, using an in-gel kinase assay with HSP105alpha or HSP105beta as the substrate, the protein kinase that phosphorylates HSP105alpha and HSP105beta was identified as casein kinase II. Since phosphorylated HSP105alpha is especially prominent in the brain compared to other tissues of mice and rats, the phosphorylation of HSP105alpha by casein kinase II may be biologically significant.     

Shp2 is required for protein kinase C-dependent phosphorylation of serine 307 in insulin receptor substrate-1

The function of insulin receptor substrate-1 (IRS-1), a key molecule of insulin signaling, is modulated by phosphorylation at multiple serine/threonine residues. Phorbol ester stimulation of cells induces phosphorylation of two inhibitory serine residues in IRS-1, i.e. Ser-307 and Ser-318, suggesting that both sites may be targets of protein kinase C (PKC) isoforms. However, in an in vitro system using a broad spectrum of PKC isoforms (alpha, beta1, beta2, delta, epsilon, eta, mu), we detected only Ser-318, but not Ser-307 phosphorylation, suggesting that phorbol ester-induced phosphorylation of this site in intact cells requires additional signaling elements and serine kinases that link PKC activation to Ser-307 phosphorylation. As we have observed recently that the tyrosine phosphatase Shp2, a negative regulator of insulin signaling, is a substrate of PKC, we studied the role of Shp2 in this context. We found that phorbol ester-induced Ser-307 phosphorylation is reduced markedly in Shp2-deficient mouse embryonic fibroblasts (Shp2-/-) whereas Ser-318 phosphorylation is unaltered. The Ser-307 phosphorylation was rescued by transfection of mouse embryonic fibroblasts with wild-type Shp2 or with a phosphatase-inactive Shp2 mutant, respectively. In this cell model, tumor necrosis factor-alpha-induced Ser-307 phosphorylation as well depended on the presence of Shp2. Furthermore, Shp2-dependent phorbol ester effects on Ser-307 were blocked by wortmannin, rapamycin, and the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125. This suggests an involvement of the phosphatidylinositol 3-kinase/mammalian target of rapamycin cascade and of JNK in this signaling pathway resulting in IRS-1 Ser-307 phosphorylation. Because the activation of these kinases does not depend on Shp2, it is concluded that the function of Shp2 is to direct these activated kinases to IRS-1.     

Requirement for 3-phosphoinositide-kependent dinase-1 (PDK-1) in insulin-induced glucose uptake in immortalized brown adipocytes

To provide insight into the physiological importance of 3-phosphoinositide-dependent kinase-1 (PDK-1) in the metabolic actions of insulin, we have generated mice that harbor a PDK-1 gene containing LoxP sites (PDK-1(lox/lox) mice) and established immortalized brown preadipocyte cell lines both from these animals and from wild-type mice. Exposure to appropriate hormonal inducers resulted in the differentiation of >80% of the immortalized brown preadipocytes derived from both types of mice into mature adipocytes. Introduction of the Cre recombinase with the use of adenovirus-mediated gene transfer induced a dose-dependent decrease in the abundance of PDK-1 in PDK-1(lox/lox) adipocytes but not in the wild-type cells. In Cre-expressing PDK-1(lox/lox) adipocytes in which the abundance of PDK-1 was reduced by approximately 85%, the insulin-induced phosphorylation both of Akt on threonine 308 and of p70 S6 kinase on threonine-389 was markedly inhibited. The phosphorylation both of Akt on serine 473 and of p42 and p44 isoforms of mitogen-activated protein kinase induced by insulin was not affected by Cre expression, indicating that the latter specifically inhibits PDK-1-dependent signaling. Both glucose uptake and the translocation of glucose transporter 4 to the plasma membrane induced by insulin as well as glucose uptake induced by a constitutively active form of phosphoinositide 3-kinase were also greatly inhibited by Cre expression in PDK-1(lox/lox) adipocytes. Phosphorylation of AMP-activated protein kinase and glucose uptake induced by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) were not affected by Cre expression in PDK-1(lox/lox) adipocytes. These results indicate that PDK-1 is essential for insulin-induced glucose uptake in adipocytes.     

Signal transduction by the alpha 6 beta 4 integrin: distinct beta 4 subunit sites mediate recruitment of Shc/Grb2 and association with the cytoskeleton of hemidesmosomes.

We have examined the mechanism of signal transduction by the hemidesmosomal integrin alpha 6 beta 4, a laminin receptor involved in morphogenesis and tumor progression. Immunoprecipitation and immune complex kinase assays indicated that antibody- or laminin-induced ligation of alpha 6 beta 4 causes tyrosine phosphorylation of the beta 4 subunit in intact cells and that this event is mediated by a protein kinase(s) physically associated with the integrin. Co-immunoprecipitation and GST fusion protein binding experiments showed that the adaptor protein Shc forms a complex with the tyrosine-phosphorylated beta 4 subunit. Shc is then phosphorylated on tyrosine residues and recruits the adaptor Grb2, thereby potentially linking alpha 6 beta 4 to the ras pathway. The beta 4 subunit was found to be phosphorylated at multiple tyrosine residues in vivo, including a tyrosine-based activation motif (TAM) resembling those found in T and B cell receptors. Phenylalanine substitutions at the beta 4 TAM disrupted association of alpha 6 beta 4 with hemidesmosomes, but did not interfere with tyrosine phosphorylation of Shc and recruitment of Grb2. These results indicate that signal transduction by the alpha 6 beta 4 integrin is mediated by an associated tyrosine kinase and that phosphorylation of distinct sites in the beta 4 tail mediates assembly of the hemidesmosomal cytoskeleton and recruitment of Shc/Grb2.     

Phospholipase C-gamma 1 and phospholipase C-gamma 2 are substrates of the B cell antigen receptor associated protein tyrosine kinase.

Cross-linking the antigen receptor on B cells results in a rapid increase in protein tyrosine kinase activity as detected by increased phosphorylation on tyrosine residues of multiple proteins. Although the identity of most of this substrates remains unknown, some have been proposed. One possible substrate of the antigen receptor-associated kinase is phospholipase C (PLC). Since multiple isoforms of PLC have been identified, we have studied which isoforms are targets of the antigen receptor. PLC-gamma 1 and PLC-gamma 2 but not PLC-beta 1 or PLC-delta 1 were detected in human B cells. Immunoprecipitation with antibodies against PLC-gamma 1 or PLC-gamma 2 and subsequent Western blotting with anti-phosphotyrosine antibodies revealed that both PLC-gamma 1 and PLC-gamma 2 are tyrosine phosphorylated in stimulated but not in resting B cells. This was confirmed by experiments whereby B cell lysates were immunoprecipitated with anti-phosphotyrosine antibody and subsequently blotted with antibodies against PLC-gamma 1 or PLC-gamma 2. Further, the specific protein tyrosine kinase inhibitors, tyrphostins, which block phospholipase-C activation and proliferation of B cells also inhibited tyrosine phosphorylation on both PLC-gamma 1 and PLC-gamma 2. We conclude that both isoforms PLC-gamma 1 and PLC-gamma 2 are targets of the antigen receptor-associated protein tyrosine kinase.     

Isolation and characterization of mouse testis specific serine/threonine kinase 5 possessing four alternatively spliced variants

Phosphorylation on serine/threonine or tyrosine residues of target proteins is an essential and significant regulatory mechanism in signal transduction during many cellular and life processes, including spermatogenesis, oogenesis and fertilization. In the present work, we reported the isolation and characterization of mouse testis-specific serine/threonine kinase 5 (Tssk5), which contains four alternatively spliced variants including, Tssk5alpha, Tssk5beta, Tssk5gamma and Tssk5delta. Moreover, the locus of Tssk5 is on chromosome 14qC3 and the four variants had a similar high expression in the testis and the heart; however, had a low expression in other tissues, except for Tssk5alpha which also had comparably high expression in the spleen. Each variant of Tssk5 expression began in the testis 16 days after birth. Aside from TSSK5alpha, the other isoforms have an insertion of ten amino acid residues (RLTPSLSAAG) in region VIb (HRD domain) (His-Arg-Asp). Moreover, only TSSK5alpha exhibited kinase activity and consistently, a further Luciferase Reporter Assay demonstrated that TSSK5beta, TSSK5gamma and TSSK5delta cannot be stimulated at the CREB/CRE responsive pathway in cmparison to TSSK5alpha. These findings suggest that TSSK5beta, TSSK5gamma, TSSK5delta may be pseudokinases due to the insertion, which may damage the structure responsible for active kinase activity. Pull-down assay experiments indicated that TSSK5beta, TSSK5gamma and TSSK5delta can directly interact with TSSK5alpha. In summary, these four isoforms with similar expression patterns may be involved in spermatogenesis through a coordinative way in testis.     

Phosphorylation of the catalytic subunit of protein kinase A. Autophosphorylation versus phosphorylation by phosphoinositide-dependent kinase-1

The identification of phosphoinositide-dependent kinase-1 (PDK-1) as an activating kinase for members of the AGC family of kinases has led to its implication as the activating kinase for cAMP-dependent protein kinase. It has been established in vitro that PDK-1 can phosphorylate the catalytic (C) subunit (), but the Escherichia coli-expressed C-subunit undergoes autophosphorylation. To assess which of these mechanisms occurs in mammalian cells, a set of mutations was engineered flanking the site of PDK-1 phosphorylation, Thr-197, on the activation segment of the C-subunit. Two distinct requirements appeared for autophosphorylation and phosphorylation by PDK-1. Autophosphorylation was disrupted by mutations that compromised activity (Thr-201 and Gly-200) or altered substrate recognition (Arg-194). Conversely, only residues peripheral to Thr-197 altered PDK-1 phosphorylation, including a potential hydrophobic PDK-1 binding site at the C terminus. To address the in vivo requirements for phosphorylation, select mutant proteins were transfected into COS-7 cells, and their phosphorylation state was assessed with phospho-specific antibodies. The phosphorylation pattern of these mutant proteins indicates that autophosphorylation is not the maturation mechanism in the eukaryotic cell; instead, a heterologous kinase with properties resembling the in vitro characteristics of PDK-1 is responsible for in vivo phosphorylation of PKA.     

Membrane localization of 3-phosphoinositide-dependent protein kinase-1 stimulates activities of Akt and atypical protein kinase C but does not stimulate glucose transport and glycogen synthesis in 3T3-L1 adipocytes

It is reported that 3-phosphoinositide-dependent protein kinase-1 (PDK-1) is activated in a phosphatidylinositol 3,4,5-trisphosphate-dependent manner and phosphorylates Akt, p70S6 kinase, and atypical protein kinase C (PKC), but its function on insulin signaling is still unclear. We cloned a full-length pdk-1 cDNA from a human brain cDNA library, and the adenovirus to overexpress wild type PDK-1 (PDK-1WT) or membrane-targeted PDK-1 (PDK-1CAAX) was constructed. Overexpressed PDK-1WT existed mainly at cytosol, and PDK-1CAAX was located at the plasma membrane. In 3T3-L1 adipocytes, insulin induced mobility shift of PDK-1 protein, but overexpressed PDK-1WT and CAAX were shifted at the basal state. Insulin stimulated tyrosine phosphorylation of PDK-1WT, but PDK-1CAAX was already tyrosine-phosphorylated at the basal state. Overexpression of PDK-1WT led to a full activation of PKC zeta/lambda without insulin stimulation but showed only the minimum effects to stimulate phosphorylation of Akt and GSK-3. In contrast, the overexpression of PDK-1CAAX caused phosphorylation of Akt and GSK-3 more strongly without insulin stimulation. However, PDK-1CAAX did not affect 2-deoxyglucose uptake and inhibited glycogen synthesis, surprisingly. Finally, PDK-1CAAX expression inhibited insulin-induced ERK1/2 phosphorylation in a dose-dependent manner. Taken together, the translocation of PDK-1 from cytosol to the plasma membrane is critical for Akt and GSK-3 activation. On the other hand, only atypical PKC and Akt activation was insufficient for stimulation of glucose transport, and constitutive activation of Akt-GSK-3 pathway may inhibit glycogen synthesis and MAPK cascade in 3T3-L1 adipocytes.     

A biochemical analysis of topoisomerase II alpha and beta kinase activity found in HIV-1 infected cells and virus

Human topoisomerase II plays a crucial role in DNA replication and repair. It exists in two isoforms: topoisomerase II alpha (alpha) and topoisomerase II beta (beta). The alpha isoform is localized predominantly in the nucleus, while the beta isoform exhibits a reticular pattern of distribution both in the cytosol and in the nucleus. We show that both isoforms of topoisomerase II are phosphorylated in HIV infected cells and also by purified viral lysate. An analysis of the phosphorylation of topoisomerase II isoforms showed that extracts of HIV infected cells at 8 and 32 h. post-infection (p.i.) contain maximal phosphorylated topoisomerase II alpha, whereas infected cell extracts at 4 and 64 h p.i. contain maximum levels of phosphorylated topoisomerase II beta. In concurrent to phosphorylated topoisomerase II isoforms, we have also observed increased topoisomerase II alpha kinase activity after 8h p.i and topoisomerase beta kinase activity at 4 and 64 h p.i. These findings suggest that both topoisomerase II alpha and beta kinase activities play an important role in early as well as late stages of HIV-1 replication. Further analysis of purified virus showed that HIV-1 virion contained topoisomerase II isoform-specific kinase activities, which were partially isolated. One of the kinase activities of higher hydrophobicity can phosphorylate both topoisomerase II alpha and beta, while lower hydrophobic kinase could predominantly phosphorylate topoisomerase II alpha. The phosphorylation status was correlated with catalytic activity of the enzyme. Western blot analysis using phosphoamino-specific antibodies shows that both the kinase activities catalyze the phosphorylation at serine residues of topoisomerase II alpha and beta. The catalytic inhibitions by serine kinase inhibitors further suggest that the alpha and beta kinase activities associated with virus are distinctly different.     

Five isoforms of the phosphatidylinositol 3-kinase regulatory subunit exhibit different associations with receptor tyrosine kinases and their tyrosine phosphorylations

There are five isoforms of the regulatory subunit for the heterodimeric type of phosphatidylinositol 3-kinase. These five regulatory subunit isoforms were overexpressed using an adenovirus transfection system, and their own tyrosine phosphorylations and associations with various tyrosine kinase receptors were investigated. When overexpressed in CHO-PDGFR cells, the associations of these regulatory subunit isoforms with the platelet-derived growth factor receptor were similar. However, when overexpressed in CHO-IR cells, p55gamma exhibited a significantly lower ability to bind with IRS-1 upon insulin stimulation, as compared with other regulatory subunit isoforms. Furthermore, p55alpha and p55gamma were found to be tyrosine-phosphorylated. Finally, interestingly, when overexpressed in CHO-EGFR cells or A431 cells and stimulated with epidermal growth factor (EGF), phosphorylated EGF receptor was detected in p85alpha, p85beta and p50alpha immunoprecipitates, but not in p55alpha and p55gamma immunoprecipitates. In addition, EGF-induced tyrosine phosphorylation was observed in p85alpha, p85beta, p55alpha and p55gamma, but not in p50alpha, immunoprecipitates. Thus, each regulatory subunit exhibits specific responses regarding both the association with tyrosine-phosphorylated substrates and its own tyrosine phosphorylation. These results suggest that each isoform possesses specific roles in signal transduction, based on its individual tyrosine kinase receptor.     

Tyrosine phosphorylation of phosphoinositide-dependent kinase 1 by the insulin receptor is necessary for insulin metabolic signaling

In L6 myoblasts, insulin receptors with deletion of the C-terminal 43 amino acids (IR(Delta43)) exhibited normal autophosphorylation and IRS-1/2 tyrosine phosphorylation. The L6 cells expressing IR(Delta43) (L6(IRDelta43)) also showed no insulin effect on glucose uptake and glycogen synthase, accompanied by a >80% decrease in insulin induction of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) activity and tyrosine phosphorylation and of protein kinase B (PKB) phosphorylation at Thr(308). Insulin induced the phosphatidylinositol 3 kinase-dependent coprecipitation of PDK-1 with wild-type IR (IR(WT)), but not IR(Delta43). Based on overlay blotting, PDK-1 directly bound IR(WT), but not IR(Delta43). Insulin-activated IR(WT), and not IR(Delta43), phosphorylated PDK-1 at tyrosines 9, 373, and 376. The IR C-terminal 43-amino-acid peptide (C-terminal peptide) inhibited in vitro PDK-1 tyrosine phosphorylation by the IR. Tyr-->Phe substitution prevented this inhibitory action. In the L6(hIR) cells, the C-terminal peptide coprecipitated with PDK-1 in an insulin-stimulated fashion. This peptide simultaneously impaired the insulin effect on PDK-1 coprecipitation with IR(WT), on PDK-1 tyrosine phosphorylation, on PKB phosphorylation at Thr(308), and on glucose uptake. Upon insulin exposure, PDK-1 membrane persistence was significantly reduced in L6(IRDelta43) compared to control cells. In L6 cells expressing IR(WT), the C-terminal peptide also impaired insulin-dependent PDK-1 membrane persistence. Thus, PDK-1 directly binds to the insulin receptor, followed by PDK-1 activation and insulin metabolic effects.     

Loss of PTEN expression does not contribute to PDK-1 activity and PKC activation-loop phosphorylation in Jurkat leukaemic T cells

Unopposed PI3-kinase activity and 3'-phosphoinositide production in Jurkat T cells, due to a mutation in the PTEN tumour suppressor protein, results in deregulation of PH domain-containing proteins including the serine/threonine kinase PKB/Akt. In Jurkat cells, PKB/Akt is constitutively active and phosphorylated at the activation-loop residue (Thr308). 3'-phosphoinositide-dependent protein kinase-1 (PDK-1), an enzyme that also contains a PH domain, is thought to catalyse Thr308 phosphorylation of PKB/Akt in addition to other kinase families such as PKC isoforms. It is unknown however if the loss of PTEN in Jurkat cells also results in unregulated PDK-1 activity and whether such loss impacts on activation-loop phosphorylation of other putative PDK-1 substrates such as PKC. In this study we have addressed if loss of PTEN in Jurkat T cells affects PDK-1 catalytic activity and intracellular localisation. We demonstrate that reducing the level of 3'-phosphoinositides in Jurkat cells with pharmacological inhibitors of PI3-kinase or expression of PTEN does not affect PDK-1 activity, Ser241 phosphorylation or intracellular localisation. In support of this finding, we show that the levels of PKC activation-loop phosphorylation are unaffected by reductions in the levels of 3'-phosphoinositides. Instead, the dephosphorylation that occurs on PKB/Akt at Thr308 following reductions in 3'-phosphoinositides is dependent on PP2A-like phosphatase activity. Our finding that PDK-1 functions independently of 3'-phosphoinositides in T cells is also confirmed by studies in HuT-78 T cells, a PTEN-expressing cell line with undetectable levels of 3'-phosphoinositides. We conclude therefore that loss of PTEN expression in Jurkat T cells does not impact on the PDK-1/PKC pathway and that only a subset of kinases, such as PKB/Akt, are perturbed as a consequence PTEN loss.     

Fibroblast growth factor receptor 1 (FGFR1) tyrosine phosphorylation regulates binding of FGFR substrate 2alpha (FRS2alpha) but not FRS2 to the receptor

Binding of the fibroblast growth factor (FGF) to the FGF receptor (FGFR) tyrosine kinase leads to receptor tyrosine autophosphorylation as well as phosphorylation of multiple downstream signaling molecules that are recruited to the receptor either by direct binding or through adaptor proteins. The FGFR substrate 2 (FRS2) family consists of two members, FRS2alpha and FRS2beta, and has been shown to recruit multiple signaling molecules, including Grb2 and Shp2, to FGFR1. To better understand how FRS2 interacted with FGFR1, in vivo binding assays with coexpressed FGFR1 and FRS2 recombinant proteins in mammalian cells were carried out. The results showed that the interaction of full-length FRS2alpha, but not FRS2beta, with FGFR1 was enhanced by activation of the receptor kinase. The truncated FRS2alpha mutant that was comprised only of the phosphotyrosine-binding domain (PTB) bound FGFR1 constitutively, suggesting that the C-terminal sequence downstream the PTB domain inhibited the PTB-FGFR1 binding. Inactivation of the FGFR1 kinase and substitutions of tyrosine phosphorylation sites of FGFR1, but not FRS2alpha, reduced binding of FGFR1 with FRS2alpha. The results suggest that although the tyrosine autophosphorylation sites of FGFR1 did not constitute the binding sites for FRS2alpha, phosphorylation of these residues was essential for optimal interaction with FRS2alpha. In addition, it was demonstrated that the Grb2-binding sites of FRS2alpha are essential for mediating signals of FGFR1 to activate the FiRE enhancer of the mouse syndecan 1 gene. The results, for the first time, demonstrate the specific signals mediated by the Grb2-binding sites and further our understanding of FGF signal transmission at the adaptor level.     

IL-2 regulation of tyrosine kinase activity is mediated through the p70-75 beta-subunit of the IL-2 receptor

The stimulation of activated human T lymphocytes with IL-2 results in increased tyrosine kinase activity. IL-2 treatment of Tac+ T cells stimulates the rapid phosphorylation of multiple protein substrates at M of 116, 100, 92, 70 to 75, 60, 56, 55, 33, and 32 kDa. Phosphorylation on tyrosine residues was detected by immunoaffinity purification of protein substrates with Sepharose linked antiphosphotyrosine mAb, 1G2. Although phorbol ester stimulated serine phosphorylation of the IL-2R alpha (p55) subunit recognized by alpha TAC mAb, IL-2 did not stimulate any detectable phosphorylation of IL-2R alpha or associated coimmune precipitated proteins. In fact, the tyrosine phosphorylated proteins did not coprecipitate with alpha Tac antibody and similar phosphoproteins were stimulated by IL-2 in IL-2R alpha- human large granular lymphocytes which express only the 70 to 75 kDa IL-2R beta subunit of the high affinity IL-2R. Anti-Tac mAb could inhibit IL-2-stimulated tyrosine phosphorylation in activated T cells, which express both IL-2R subunits that together form the high affinity receptor complex, but not in large granular lymphocytes expressing only the IL-2R beta subunit. The data suggest that IL-2 stimulation of tyrosine kinase activities requires only the IL-2R beta subunit.     

A tyrosine kinase physically associates with the beta-subunit of the human IL-2 receptor

Cell surface expression of the high affinity IL-2R regulates, in part, the proliferative response occurring in Ag- or mitogen-activated T cells. The functional high affinity IL-2R is composed of at least two distinct ligand-binding components, IL-2R alpha (Tac, p55) and IL-2R beta (p70/75). The IL-2R beta polypeptide appears to be essential for growth signal transduction, whereas the IL-2R alpha protein participates in the regulation of receptor affinity. We have prepared and characterized two mAb, DU-1 and DU-2, that specifically react with IL-2R beta. In vitro kinase assays performed with DU-2 immunoprecipitates, but not anti-IL-2R alpha or control antibody immunoprecipitates, have revealed co-precipitation of a tyrosine kinase enzymatic activity that mediates phosphorylation of IL-2R beta. Because both IL-2R alpha and IL-2R beta lack tyrosine kinase enzymatic domains, these findings strongly suggest that noncovalent association of a tyrosine kinase with the high affinity IL-2R complex. Deletion mutants of the intracellular region of IL-2R beta, lacking either a previously described "critical domain" between amino acids 267 and 322 or the carboxyl-terminal 198 residues (IL-2R beta 88), lacked the ability to co-precipitate this tyrosine kinase activity, as measured by phosphorylation of IL-2R beta in vitro. Both of these mutants also failed to transduce growth-promoting signals in response to IL-2 in vivo. Analysis of the IL-2R beta 88 mutant receptor suggested that a second protein kinase mediating phosphorylation on serine and threonine residues physically interacts with the carboxyl terminus of IL-2R beta. This kinase may be necessary but, alone, appears to be insufficient to support a full IL-2-induced proliferative response. These studies highlight the physical association of protein kinases with the cytoplasmic domain of IL-2R beta and their likely role in IL-2-induced growth signaling mediated through the multimeric high affinity IL-2R complex.