FER kinase activation of Stat3 is determined by the N-terminal sequence

p94(fer) and p51(ferT) are two tyrosine kinases that share identical SH2 and kinase domains but differ in their N-terminal regions. To further explore the cellular functions of these two highly related tyrosine kinases, their subcellular distribution profiles and in vivo phosphorylation activity were followed using double immunofluorescence assay. When combined with immunoprecipitation analysis, this assay showed that p94(fer) can lead to the tyrosine phosphorylation and activation of Stat3 but not of Stat1 or Stat2. Native p94(fer) exerted this activity when residing in the cytoplasm. However, modified forms of p94(fer), which are constitutively nuclear, could also lead to the phosphorylation of Stat3. Endogenous Stat3 and p94(fer) co-immunoprecipitated with each other, thus proving the interaction of these two proteins in vivo. Unlike p94(fer), p51(ferT) did not induce the phosphorylation of Stat3 but led to the phosphorylation of other nuclear proteins. Replacing the unique 43-amino acid-long N-terminal tail of p51(ferT) with a parallel segment from the N-terminal tail of p94(fer) did not change the subcellular localization of p51(ferT) but enabled it to activate Stat3. Thus the different N-terminal sequences of p94(fer) and p51(ferT) can affect their ability to induce phosphorylation of Stat3 and most probably direct their different cellular functions.     

N-terminal sequences direct the autophosphorylation states of the FER tyrosine kinases in vivo

p94(fer) and p51(ferT) are two tyrosine kinases which share identical SH2 and kinase domains but differ in their N-terminal regions. While p94(fer) is expressed in most mammalian cells, the accumulation of p51(ferT) is restricted to meiotic spermatocytes. Here we show that the different N-terminal tails of p94(fer) and p51(ferT) direct different autophosphorylation states of these two kinases in vivo. N-terminal coiled-coil domains cooperated to drive the oligomerization and autophosphorylation in trans of p94(fer). Moreover, the ectopically expressed N-terminal tail of p94(fer) could act as a dominant negative mutant and associated with the endogenous p94(fer) protein in CHO cells. This increased significantly the percentage of cells residing in the G0/G1 phase, thus suggesting a role for p94(fer) in the regulation of G1 progression. Unlike p94(fer), overexpressed p51(ferT) was not autophosphorylated in COS1 cells. However, removal of the unique N-terminal 43 aa of p51(ferT) or the replacement of this region by a parallel segment from p94(fer) endowed the modified p51(ferT) with the ability to autophosphorylate. The unique N-terminal sequences of p51(ferT) thus interfere with its ability to autophosphorylate in vivo. These experiments indicate that the N-terminal sequences of the FER tyrosine kinases direct their different cellular autophosphorylation states, thereby dictating their different cellular functions.     

Nuclear localization and apoptotic regulation of an amino-terminal domain focal adhesion kinase fragment in endothelial cells

This study investigated the subcellular compartmentalization of focal adhesion kinase (FAK) fragments and their regulation during apoptosis of human umbilical vein endothelial cells. A 50 kDa NH(2)-terminal FAK fragment and a 120 kDa FAK variant were constitutively expressed and specifically found in the nuclear fraction of cells, while a 55 kDa COOH-terminal FAK fragment was only in the cytosolic fraction. FAK cleavage fragments generated during apoptosisremained in the cytosol, while p120FAK and p50 NH(2)-terminal FAK remained in the nuclear compartment. The caspase inhibitor, ZVAD-fmk, prevented the apoptosis-induced proteolysis of p125 and p120FAK, generation of the 80 kDa cleavage product, and increased expression of p50N-FAK. Western blot with phospho-specific FAK showed that nuclear p125(FAK) was phosphorylated at a significant level at Y861, while FAK phosphorylated at Y397 and Y407 was largely in the cytosol. These results indicate that FAK NH(2)- and COOH-terminal domain fragments are segregated between nuclear and cytosolic compartments in endothelial cells and suggest novel functions for the FAK NH(2)-terminal domain.     

The cytoplasmic tyrosine kinase FER is associated with the catenin-like substrate pp120 and is activated by growth factors.

The FER gene encodes a cytoplasmic tyrosine kinase with a single SH2 domain and an extensive amino terminus. In order to understand the cellular function of the FER kinase, we analyzed the effect of growth factor stimulation on the phosphorylation and activity of FER. Stimulation of A431 cells and 3T3 fibroblasts with epidermal growth factor or platelet-derived growth factor results in the phosphorylation of FER and two associated polypeptides. The associated polypeptides were shown to be the epidermal growth factor receptor or the platelet-derived growth factor receptor and a previously identified target, pp120. Since pp120 had previously been shown to interact with components of the cadherin-catenin complex, these results implicate FER in the regulation of cell-cell interactions. The physical association of FER with pp120 was found to be constitutive and was mediated by a 400-amino-acid sequence in the amino terminus of FER. Analyses of that sequence revealed that it has the ability to form coiled coils and that it oligomerizes in vitro. The identification of a coiled coil sequence in the FER kinase and the demonstration that the sequence mediates association with a potential substrate suggest a novel mechanism for signal transduction by cytoplasmic tyrosine kinases.     

Identification and characterization of a cytosolic protein tyrosine kinase of HeLa cells.

Fractionation of a cytosolic extract of HeLa cells revealed the existence of a highly active protein tyrosine kinase. Chromatographic fractionation of the extract resulted in partial purification of a single enzymatic activity that coeluted with a 94-kDa polypeptide. In vitro phosphorylation of the isolated enzyme showed that p94 was the only polypeptide phosphorylated and only the tyrosine residue(s) was (were) modified. The fractionated enzyme (p94 kinase) also phosphorylated a number of other nonspecific substrates exclusively on tyrosine residues. Unlike other protein tyrosine kinases that have been characterized, p94 kinase is relatively insensitive to inhibition by the isoflavone genistein. Using two different antisera, we provided evidence that the HeLa p94 kinase is most likely the FER gene product, which was previously shown to be expressed in a wide variety of cell types. These results represent the first biochemical characterization of the cellular FER gene product and also provide a basis for studying the biochemistry of tyrosine kinase function in HeLa cells.     

Signal transduction within the nucleus by mitogen-activated protein kinase.

The nucleus is an important target of signal transduction by growth factor receptors that stimulate mitogen-activated protein (MAP) kinases. We tested the hypothesis that MAP kinases have a signaling role within the nucleus by examining the effect of the expression of a human MAP kinase isoform (p41mapk) in tissue culture cells. The expressed p41mapk was found to be localized in both the cytoplasmic and nuclear compartments of the cells. Significantly, the expression of p41mapk caused an increase in the phosphorylation of a nuclear substrate: Ser62 of c-Myc. Phosphorylation at Ser62 stimulated the activity of the NH2-terminal transactivation domain of c-Myc. Thus, p41mapk causes the phosphorylation and regulation of a physiologically significant nuclear target of signal transduction. These data establish that at least one MAP kinase isoform has a nuclear role during signal transduction.     

Interplay between kinase domain autophosphorylation and F-actin binding domain in regulating imatinib sensitivity and nuclear import of BCR-ABL

Background

The constitutively activated BCR-ABL tyrosine kinase of chronic myeloid leukemia (CML) is localized exclusively to the cytoplasm despite the three nuclear localization signals (NLS) in the ABL portion of this fusion protein. The NLS function of BCR-ABL is re-activated by a kinase inhibitor, imatinib, and in a kinase-defective BCR-ABL mutant. The mechanism of this kinase-dependent inhibition of the NLS function is not understood.

Methodology/Principal Findings

By examining the subcellular localization of mutant BCR-ABL proteins under conditions of imatinib and/or leptomycin B treatment to inhibit nuclear export, we have found that mutations of three specific tyrosines (Y232, Y253, Y257, according to ABL-1a numbering) in the kinase domain can inhibit the NLS function of kinase-proficient and kinase-defective BCR-ABL. Interestingly, binding of imatinib to the kinase-defective tyrosine-mutant restored the NLS function, suggesting that the kinase domain conformation induced by imatinib-binding is critical to the re-activation of the NLS function. The C-terminal region of ABL contains an F-actin binding domain (FABD). We examined the subcellular localization of several FABD-mutants and found that this domain is also required for the activated kinase to inhibit the NLS function; however, the binding to F-actin per se is not important. Furthermore, we found that some of the C-terminal deletions reduced the kinase sensitivity to imatinib.

Conclusions/Significance

Results from this study suggest that an autophosphorylation-dependent kinase conformation together with the C-terminal region including the FABD imposes a blockade of the BCR-ABL NLS function. Conversely, conformation of the C-terminal region including the FABD can influence the binding affinity of imatinib for the kinase domain. Elucidating the structural interactions among the kinase domain, the NLS region and the FABD may therefore provide insights on the design of next generation BCR-ABL inhibitors for the treatment of CML.     

PAKa, a putative PAK family member, is required for cytokinesis and the regulation of the cytoskeleton in Dictyostelium discoideum cells during chemotaxis.

We have identified a Dictyostelium discoideum gene encoding a serine/threonine kinase, PAKa, a putative member of the Ste20/PAK family of p21-activated kinases, with a kinase domain and a long NH(2)-terminal regulatory domain containing an acidic segment, a polyproline domain, and a CRIB domain. PAKa colocalizes with myosin II to the cleavage furrow of dividing cells and the posterior of polarized, chemotaxing cells via its NH(2)-terminal domain. paka null cells are defective in completing cytokinesis in suspension. PAKa is also required for maintaining the direction of cell movement, suppressing lateral pseudopod extension, and proper retraction of the posterior of chemotaxing cells. paka null cells are defective in myosin II assembly, as the myosin II cap in the posterior of chemotaxing cells and myosin II assembly into cytoskeleton upon cAMP stimulation are absent in these cells, while constitutively active PAKa leads to an upregulation of myosin II assembly. PAKa kinase activity against histone 2B is transiently stimulated and PAKa incorporates into the cytoskeleton with kinetics similar to those of myosin II assembly in response to chemoattractant signaling. However, PAKa does not phosphorylate myosin II. We suggest that PAKa is a major regulator of myosin II assembly, but does so by negatively regulating myosin II heavy chain kinase.     

Downregulation of the Ras-mitogen-activated protein kinase pathway by the EphB2 receptor tyrosine kinase is required for ephrin-induced neurite retraction

Activation of the EphB2 receptor tyrosine kinase by clustered ephrin-B1 induces growth cone collapse and neurite retraction in differentiated NG108 neuronal cells. We have investigated the cytoplasmic signaling events associated with EphB2-induced cytoskeletal reorganization in these neuronal cells. We find that unlike other receptor tyrosine kinases, EphB2 induces a pronounced downregulation of GTP-bound Ras and consequently of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. A similar inhibition of the Ras-MAPK pathway was observed on stimulation of endogenous EphB2 in COS-1 cells. Inactivation of Ras, induced by ephrin B1 stimulation of NG108 neuronal cells, requires EphB2 tyrosine kinase activity and is blocked by a truncated form of p120-Ras GTPase-activating protein (p120-RasGAP), suggesting that EphB2 signals through the SH2 domain protein p120-RasGAP to inhibit the Ras-MAPK pathway. Suppression of Ras activity appears functionally important, since expression of a constitutively active variant of Ras impaired the ability of EphB2 to induce neurite retraction. In addition, EphB2 attenuated the elevation in ERK activation induced by attachment of NG108 cells to fibronectin, indicating that the EphB2 receptor can modulate integrin signaling to the Ras GTPase. These results suggest that a primary function of EphB2, a member of the most populous family of receptor tyrosine kinases, is to inactivate the Ras-MAPK pathway in a fashion that contributes to cytoskeletal reorganization and adhesion responses in neuronal growth cones.     

Binding of JNK/SAPK to MEKK1 is regulated by phosphorylation

We sought to characterize the role of upstream kinases in the regulation of the MAP3 kinase MEKK1 and the potential impact on signaling to MAP kinase cascades. We find that the MAP4 kinase PAK1 phosphorylates the amino terminus of MEKK1 on serine 67. We show that serine 67 lies in a D domain, which binds to the c-Jun-NH(2)-terminal kinase/stress-activated protein kinases (JNK/SAPK). Serine 67 is constitutively phosphorylated in resting 293 cells, but is dephosphorylated following exposure to stress stimuli such as anisomycin and UV irradiation. Phosphorylation of this site inhibits binding of JNK/SAPK to MEKK1. Thus, we propose a mechanism by which the MEKK1-dependent JNK/SAPK pathway is negatively regulated by PAK through phosphorylation of serine 67.     

The NPK1 mitogen-activated protein kinase kinase kinase contains a functional nuclear localization signal at the binding site for the NACK1 kinesin-like protein

The tobacco mitogen-activated protein kinase kinase kinase NPK1 localizes to the equatorial region of phragmoplasts by interacting with kinesin-like protein NACK1. This leads to activation of NPK1 kinase at late M phase, which is necessary for cell plate formation. Until now, its localization during interphase has not been reported. We investigated the subcellular localization of NPK1 in tobacco-cultured BY-2 cells at interphase using indirect immunofluorescence microscopy and fusion to green fluorescent protein (GFP). Fluorescence of anti-NPK1 antibodies and GFP-fused NPK1 were detected only in the nuclei of BY-2 cells at interphase. Examination of the amino acid sequence of NPK1 showed that at the carboxyl-terminal region in the regulatory domain, which contains the binding site of NACK1, NPK1 contained a cluster of basic amino acids that resemble a bipartite nuclear localization signal (NLS). Amino acid substitution mutations in the critical residues in putative NLS caused a marked reduction in nuclear localization of NPK1 in BY-2 cells, indicating that this sequence is functional in tobacco BY-2 cells. We also found that the 64-amino acid sequence at the carboxyl terminus that contains NLS sequence is essential for interaction with NACK1, and that mutations in the NLS sequence prevented NPK1 from interacting with NACK1. Thus, the amino acid sequence at the carboxyl-terminal region of NPK1 has dual functions for nuclear localization during interphase and binding NACK1 in M phase.     

Meiosis-dependent tyrosine phosphorylation of a yeast protein related to the mouse p51ferT

The FER locus of the mouse encodes two mRNA species: one is constitutively transcribed, giving rise to a 94 kDa tyrosine kinase (p94^ferT); the second is a meiosis-specific RNA that gives rise to a 51 kDa tyrosine kinase (p51^ferT). The p51^ferT RNA and protein accumulate in primary spermatocytes that are in prophase of the first meiotic division. By using polyclonal antibodies directed against synthetic peptides derived from the unique amino-terminus of the mouse p51^ferT, a 51 kDa phosphotyrosyl protein — p51^y — was identified in Saccharomyces cerevisiae. The p51^y protein is constitutively expressed in yeast, but in meiotic cells, concomitantly with commitment to meiotic recombination, its level of phosphorylation on tyrosine residues is increased. A different pattern of phosphorylation is observed on serine residues: at early meiotic times the level is decreased, while in later meiotic time the level increases, reaching the vegetative level. When p51^ferT is ectopically expressed in yeast, it is active, leading to preferential phosphorylation of an approx. 65 kDa protein. A similar pattern of phosphorylation by p51^ferT is seen in mammalian cells.     

Structure of mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 suggests a bifunctional switch that couples kinase activation with nuclear export

MAPK-activated protein kinase 2 (MAPKAPK2), one of several kinases directly phosphorylated and activated by p38 MAPK, plays a central role in the inflammatory response. The activated MAPKAPK2 phosphorylates its nuclear targets CREB/ATF1, serum response factor, and E2A protein E47 and its cytoplasmic targets HSP25/27, LSP-1, 5-lipoxygenase, glycogen synthase, and tyrosine hydroxylase. The crystal structure of unphosphorylated MAPKAPK2, determined at 2.8 A resolution, includes the kinase domain and the C-terminal regulatory domain. Although the protein is inactive, the kinase domain adopts an active conformation with aspartate 366 mimicking the missing phosphorylated threonine 222 in the activation loop. The C-terminal regulatory domain forms a helix-turn-helix plus a long strand. Phosphorylation of threonine 334, which is located between the kinase domain and the C-terminal regulatory domain, may serve as a switch for MAPKAPK2 nuclear import and export. Phosphorylated MAPKAPK2 masks the nuclear localization signal at its C terminus by binding to p38. It unmasks the nuclear export signal, which is part of the second C-terminal helix packed along the surface of kinase domain C-lobe, and thereby carries p38 to the cytoplasm.     

Meiosis-dependent tyrosine phosphorylation of a yeast protein related to the mouse p51ferT

The FER locus of the mouse encodes two mRNA species: one is constitutively transcribed, giving rise to a 94 kDa tyrosine kinase (p94^ferT); the second is a meiosis-specific RNA that gives rise to a 51 kDa tyrosine kinase (p51^ferT). The p51^ferT RNA and protein accumulate in primary spermatocytes that are in prophase of the first meiotic division. By using polyclonal antibodies directed against synthetic peptides derived from the unique amino-terminus of the mouse p51^ferT, a 51 kDa phosphotyrosyl protein — p51^y — was identified in Saccharomyces cerevisiae. The p51^y protein is constitutively expressed in yeast, but in meiotic cells, concomitantly with commitment to meiotic recombination, its level of phosphorylation on tyrosine residues is increased. A different pattern of phosphorylation is observed on serine residues: at early meiotic times the level is decreased, while in later meiotic time the level increases, reaching the vegetative level. When p51^ferT is ectopically expressed in yeast, it is active, leading to preferential phosphorylation of an approx. 65 kDa protein. A similar pattern of phosphorylation by p51^ferT is seen in mammalian cells.     

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.     

Caspase activation of mammalian sterile 20-like kinase 3 (Mst3). Nuclear translocation and induction of apoptosis

Mammalian Sterile 20-like kinase 3 (Mst3), the physiological functions of which are unknown, is a member of the germinal center kinase-III family. It contains a conserved kinase domain at its NH(2) terminus, whereas there is a regulatory domain at its COOH terminus. In this study we demonstrate that endogenous Mst3 is specifically cleaved when Jurkat cells were treated with anti-Fas antibody or staurosporine and that this cleavage is inhibited by the caspase inhibitor, Ac-DEVD-CHO. Using apoptotic Jurkat cell extracts and recombinant caspases, we mapped the caspase cleavage site, AETD(313), which is at the junction of the NH(2)-terminal kinase domain and the COOH-terminal regulatory domain. Caspase-mediated cleavage of Mst3 activates its intrinsic kinase activity, suggesting that the COOH-terminal domain of Mst3 negatively regulates the kinase domain. Furthermore, proteolytic removal of the Mst3 COOH-terminal domain by caspases promotes nuclear translocation. Ectopic expression of either wild-type or COOH-terminal truncated Mst3 in cells results in DNA fragmentation and morphological changes characteristic of apoptosis. By contrast, no such changes were exhibited for catalytically inactive Mst3, implicating the involvement of Mst3 kinase activity for mediation of these effects. Collectively, these results support the notion that caspase-mediated proteolytic activation of Mst3 contributes to apoptosis.