共查询到20条相似文献,搜索用时 31 毫秒
1.
2.
3.
Baljuls A Schmitz W Mueller T Zahedi RP Sickmann A Hekman M Rapp UR 《The Journal of biological chemistry》2008,283(40):27239-27254
In mammals the RAF family of serine/threonine kinases consists of three members, A-, B-, and C-RAF. Activation of RAF kinases involves a complex series of phosphorylations. Although the most prominent phosphorylation sites of B- and C-RAF are well characterized, little is known about regulatory phosphorylation of A-RAF. Using mass spectrometry, we identified here a number of novel in vivo phosphorylation sites in A-RAF. In particular, we found that Ser-432 participates in MEK binding and is indispensable for A-RAF signaling. On the other hand, phosphorylation within the activation segment does not contribute to epidermal growth factor-mediated activation. Furthermore, we show that the potential 14-3-3 binding domains in A-RAF are phosphorylated independently of its activation status. Of importance, we identified a novel regulatory domain in A-RAF (referred to as IH-segment) positioned between amino acids 248 and 267 that contains seven putative phosphorylation sites. Three of these sites, serines 257, 262, and 264, regulate A-RAF activation in a stimulatory manner. The spatial model of the A-RAF fragment, including residues between Ser-246 and Glu-277, revealed a switch of charge at the molecular surface of the IH-region upon phosphorylation, suggesting a mechanism in which the high accumulation of negative charges may lead to an electrostatic destabilization of protein-membrane interaction resulting in depletion of A-RAF from the plasma membrane. Together, we provide here for the first time a detailed analysis of in vivo A-RAF phosphorylation status and demonstrate that regulation of A-RAF by phosphorylation exhibits unique features compared with B- and C-RAF. 相似文献
4.
Abbasi S Lee JD Su B Chen X Alcon JL Yang J Kellems RE Xia Y 《The Journal of biological chemistry》2006,281(12):7717-7726
Calcineurin is a serine/threonine protein phosphatase that plays a critical role in many physiologic processes such as T-cell activation, skeletal myocyte differentiation, and cardiac hypertrophy. We previously showed that active MEKK3 is capable of stimulating calcineurin/nuclear factor of activated T-cells (NFAT) signaling in cardiac myocytes through phosphorylation of modulatory calcineurin-interacting protein 1 (MCIP1). However, the protein kinases that function downstream of MEKK3 to mediate MCIP1 phosphorylation and the mechanism of MCIP1-mediated calcineurin regulation have not been defined. Here, we show that MEK5 and big MAP kinase 1 (BMK1) function downstream of MEKK3 in a signaling cascade that induces calcineurin activity through phosphorylation of MCIP1. Genetic studies showed that BMK1-deficient mouse lung fibroblasts failed to mediate MCIP1 phosphorylation and activate calcineurin/NFAT in response to angiotensin II, a potent NFAT activator. Conversely, restoring BMK1 to the deficient cells restored angiotensin II-mediated calcineurin/NFAT activation. Thus, using BMK1-deficient mouse lung fibroblast cells, we provided the genetic evidence that BMK1 is required for angiotensin II-mediated calcineurin/NFAT activation through MICP1 phosphorylation. Finally, we discovered that phosphorylated MCIP1 dissociates from calcineurin and binds with 14-3-3, thereby relieving its inhibitory effect on calcineurin activity. In summary, our findings reveal a previously unrecognized essential regulatory role of mitogen-activated protein kinase signaling in calcineurin activation through the reversible phosphorylation of a calcineurin-interacting protein, MCIP1. 相似文献
5.
6.
7.
Structural modes of stabilization of permissive phosphorylation sites in protein kinases: distinct strategies in Ser/Thr and Tyr kinases 总被引:5,自引:0,他引:5
Protein kinases phosphorylate several cellular proteins providing control mechanisms for various signalling processes. Their activity is impeded in a number of ways and restored by alteration in their structural properties leading to a catalytically active state. Most protein kinases are subjected to positive and negative regulation by phosphorylation of Ser/Thr/Tyr residues at specific sites within and outside the catalytic core. The current review describes the analysis on 3D structures of protein kinases that revealed features distinct to active states of Ser/Thr and Tyr kinases. The nature and extent of interactions among well-conserved residues surrounding the permissive phosphorylation sites differ among the two classes of enzymes. The network of interactions of highly conserved Arg preceding the catalytic base that mediates stabilization of the activation segment exemplifies such diverse interactions in the two groups of kinases. The N-terminal and the C-terminal lobes of various groups of protein kinases further show variations in their extent of coupling as suggested from the extent of interactions between key functional residues in activation segment and the N-terminal alphaC-helix. We observe higher similarity in the conformations of ATP bound to active forms of protein kinases compared to ATP conformations in the inactive forms of kinases. The extent of structural variations accompanying phosphorylation of protein kinases is widely varied. The comparison of their crystal structures and the distinct features observed are hoped to aid in the understanding of mechanisms underlying the control of the catalytic activity of distinct subgroups of protein kinases. 相似文献
8.
Okugawa S Yanagimoto S Tsukada K Kitazawa T Koike K Kimura S Nagase H Hirai K Ota Y 《Cellular microbiology》2006,8(10):1571-1580
Flagellin, the structural component of bacterial flagella, is secreted by pathogenic and commensal bacteria, and is recognized by Toll-like receptor (TLR) 5. Flagellin is a common bacterial antigen present on most motile bacteria and is speculated to contribute to the activation of CD4+ T cells in the intestine. However, molecular mechanisms by which flagellin regulate T cell activation remains to be determined. Using Jurkat T cells or human primary T cell, we showed that flagellin stimulation induced tyrosine phosphorylation of TLR5 and activation of both mitogen-activated protein kinases and nuclear factor kappaB. In addition, stimulation by flagellin did not induce nuclear factor of activated T cells (NFAT) activation, while stimulation via the T cell receptor (TCR) leads to activation of NFAT. However, TCR-mediated NFAT activation and tyrosine phosphorylation of zeta-associated protein 70 (Zap-70) were inhibited in cells pre-stimulated by flagellin. Furthermore, flagellin stimulation induced suppressor of cytokine signalling-1 (SOCS-1), which formed a complex with Zap-70 after stimulation via TCR, and inhibition of SOCS-1 expression by SOCS-1-specific small interfering RNA reinstated TCR-mediated activation of NFAT in cells pre-stimulated with flagellin. These results collectively indicate that bacterial flagellin inhibits TCR-mediated activation of T cells by inducing SOCS-1. 相似文献
9.
10.
11.
Two distinct phosphorylation pathways have additive effects on Abl family kinase activation
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Tanis KQ Veach D Duewel HS Bornmann WG Koleske AJ 《Molecular and cellular biology》2003,23(11):3884-3896
The activities of the related Abl and Arg nonreceptor tyrosine kinases are kept under tight control in cells, but exposure to several different stimuli results in a two- to fivefold stimulation of kinase activity. Following the breakdown of inhibitory intramolecular interactions, Abl activation requires phosphorylation on several tyrosine residues, including a tyrosine in its activation loop. These activating phosphorylations have been proposed to occur either through autophosphorylation by Abl in trans or through phosphorylation of Abl by the Src nonreceptor tyrosine kinase. We show here that these two pathways mediate phosphorylation at distinct sites in Abl and Arg and have additive effects on Abl and Arg kinase activation. Abl and Arg autophosphorylate at several sites outside the activation loop, leading to 5.2- and 6.2-fold increases in kinase activity, respectively. We also find that the Src family kinase Hck phosphorylates the Abl and Arg activation loops, leading to an additional twofold stimulation of kinase activity. The autoactivation pathway may allow Abl family kinases to integrate or amplify cues relayed by Src family kinases from cell surface receptors. 相似文献
12.
13.
Humphries LA Dangelmaier C Sommer K Kipp K Kato RM Griffith N Bakman I Turk CW Daniel JL Rawlings DJ 《The Journal of biological chemistry》2004,279(36):37651-37661
Tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2) is a crucial activation switch that initiates and maintains intracellular calcium mobilization in response to B cell antigen receptor (BCR) engagement. Although members from three distinct families of non-receptor tyrosine kinases can phosphorylate PLCgamma in vitro, the specific kinase(s) controlling BCR-dependent PLCgamma activation in vivo remains unknown. Bruton's tyrosine kinase (Btk)-deficient human B cells exhibit diminished inositol 1,4,5-trisphosphate production and calcium signaling despite a normal inducible level of total PLCgamma2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLCgamma2 activity. To evaluate this hypothesis, we generated site-specific phosphotyrosine antibodies recognizing four putative regulatory residues within PLCgamma2. Whereas all four sites were rapidly modified in response to BCR engagement in normal B cells, Btk-deficient B cells exhibited a marked reduction in phosphorylation of the Src homology 2 (SH2)-SH3 linker region sites, Tyr(753) and Tyr(759). Phosphorylation of both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLCgamma2 carboxyl-terminal sites, Tyr(1197) and Tyr(1217), was unaffected by the absence of functional Btk. Together, these data support a model whereby Btk/Tec kinases control sustained calcium signaling via site-specific phosphorylation of key residues within the PLCgamma2 SH2-SH3 linker. 相似文献
14.
Pike AC Rellos P Niesen FH Turnbull A Oliver AW Parker SA Turk BE Pearl LH Knapp S 《The EMBO journal》2008,27(4):704-714
Protein kinase autophosphorylation of activation segment residues is a common regulatory mechanism in phosphorylation-dependent signalling cascades. However, the molecular mechanisms that guarantee specific and efficient phosphorylation of these sites have not been elucidated. Here, we report on three novel and diverse protein kinase structures that reveal an exchanged activation segment conformation. This dimeric arrangement results in an active kinase conformation in trans, with activation segment phosphorylation sites in close proximity to the active site of the interacting protomer. Analytical ultracentrifugation and chemical cross-linking confirmed the presence of dimers in solution. Consensus substrate sequences for each kinase showed that the identified activation segment autophosphorylation sites are non-consensus substrate sites. Based on the presented structural and functional data, a model for specific activation segment phosphorylation at non-consensus substrate sites is proposed that is likely to be common to other kinases from diverse subfamilies. 相似文献
15.
Focal adhesion kinase (FAK) is a key signaling molecule regulating cellular responses to integrin-mediated adhesion. Integrin engagement promotes FAK phosphorylation at multiple sites to achieve full FAK activation. Phosphorylation of FAK Tyr-397 creates a binding site for Src-family kinases, and phosphorylation of FAK Tyr-576/Tyr-577 in the kinase domain activation loop enhances catalytic activity. Using novel phosphospecific antibody reagents, we show that FAK activation loop phosphorylation is significantly elevated in cells expressing activated Src and is an early event following cell adhesion to fibronectin. In both cases, this regulation is largely dependent on Tyr-397. We also show that the FAK activation loop tyrosines are required for maximal Tyr-397 phosphorylation. Finally, immunostaining analyses revealed that tyrosine-phosphorylated forms of FAK are present in both newly forming and mature focal adhesions. Our findings support a model for reciprocal activation of FAK and Src-family kinases and suggest that FAK/Src signaling may occur during both focal adhesion assembly and turnover. 相似文献
16.
The hydrophobic phosphorylation motif of conventional protein kinase C is regulated by autophosphorylation. 总被引:7,自引:0,他引:7
BACKGROUND: A growing number of kinases are now known to be controlled by two phosphorylation switches, one on a loop near the entrance to the active site and a second on the carboxyl terminus. For the protein kinase C (PKC) family of enzymes, phosphorylation at the activation loop is mediated by another kinase but the mechanism for carboxy-terminal phosphorylation is still unclear. The latter switch contains two phosphorylation sites - one on a 'turn' motif and the second on a conserved hydrophobic phosphorylation motif - that are found separately or together in a number of other kinases. RESULTS: Here, we investigated whether the carboxy-terminal phosphorylation sites of a conventional PKC are controlled by autophosphorylation or by another kinase. First, kinetic analyses revealed that a purified construct of the kinase domain of PKC betaII autophosphorylated on the Ser660 residue of the hydrophobic phosphorylation motif in an apparently concentration-independent manner. Second, kinase-inactive mutants of PKC did not incorporate phosphate at either of the carboxy-terminal sites, Thr641 or Ser660, when expressed in COS-7 cells. The inability to incorporate phosphate on the hydrophobic site was unrelated to the phosphorylation state of the other key phosphorylation sites: kinase-inactive mutants with negative charge at Thr641 and/or the activation-loop position were also not phosphorylated in vivo. CONCLUSIONS: PKC betaII autophosphorylates at its conserved carboxy-terminal hydrophobic phosphorylation site by an apparently intramolecular mechanism. Expression studies with kinase-inactive mutants revealed that this mechanism is the only one responsible for phosphorylating this motif in vivo. Thus, conventional PKC autoregulates the carboxy-terminal phosphorylation switch following phosphorylation by another kinase at the activation loop switch. 相似文献
17.
18.
Florina Vlad Marie‐Jo Droillard Benoît Valot Mehdi Khafif Americo Rodrigues Mathias Brault Michel Zivy Pedro L. Rodriguez Sylvain Merlot Christiane Laurière 《The Plant journal : for cell and molecular biology》2010,63(5):778-790
Snf1‐related protein kinases 2 (SnRK2s) are major positive regulators of drought stress tolerance. The kinases of this family are activated by hyperosmotic stress, but only some of them are also responsive to abscisic acid (ABA). Moreover, genetic evidence has indicated the ABA‐independence of SnRK2 activation in the fast response to osmotic stress. Although phosphorylation was demonstrated to be crucial for the activation or activity of the kinases of both subgroups, different phosphorylation mechanisms were suggested. Here, using one kinase from each subgroup (SnRK2.6 and SnRK2.10), two phosphorylation sites within the activation loop were identified by mass spectrometry after immunoprecipitation from Arabidopsis cells treated by ABA or osmolarity. By site‐directed mutagenesis, the phosphorylation of only one of the two sites was shown to be necessary for the catalytic activity of the kinase, whereas both sites are necessary for the full activation of the two SnRK2s by hyperosmolarity or ABA. Phosphoprotein staining together with two‐dimensional PAGE followed by immunoblotting indicated distinct phosphorylation mechanisms of the two kinases. While SnRK2.6 seems to be activated through the independent phosphorylation of these two sites, a sequential process occurs in SnRK2.10, where phosphorylation of one serine is required for the phosphorylation of the other. In addition, a subgroup of protein phosphatases 2C which interact and participate in the regulation of SnRK2.6 do not interact with SnRK2.10. Taken together, our data bring evidence for the involvement of distinct phosphorylation mechanisms in the activation of SnRK2.6 and SnRK2.10, which may be conserved between the two subgroups of SnRK2s depending on their ABA‐responsiveness. 相似文献
19.
20.
A mechanism for synaptic frequency detection through autophosphorylation of CaM kinase II. 总被引:2,自引:0,他引:2
下载免费PDF全文
![点击此处可从《Biophysical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A model for the regulation of CaM kinase II is presented based on the following reported properties of the molecule: 1) The holoenzyme is composed of 8-12 subunits, each with the same set of autophosphorylation sites; 2) Autophosphorylation at one group of sites (A sites) requires the presence of Ca2+ and causes a subunit to remain active following the removal of Ca2+; 3) Autophosphorylation at another group of sites (B sites) occurs only after the removal of Ca2+ but requires prior phosphorylation of a threshold number of A sites within the holoenzyme. Because B-site phosphorylation inhibits Ca2+/calmodulin binding, we propose that, for a given subunit, phosphorylation of a B site before an A site prevents subsequent phosphorylation at the A site and thereby locks that subunit in an inactive state. The model predicts that a threshold activation by Ca2+ will initiate an "autophosphorylation phase." Once started, intra-holoenzyme autophosphorylation will proceed, on A sites during periods of high [Ca2+] and on B sites during periods of low [Ca2+]. At "saturation," that is when every subunit has been phosphorylated on a B site, the number of phosphorylated A sites and, therefore, the kinase activity will reflect the relative durations of periods of high [Ca2+] to periods of low [Ca2+] that occurred during the autophosphorylation phase. Using a computer program designed to simulate the above mechanism, we show that the ultimate state of phosphorylation of an array of CaM kinase II molecules could be sensitive to the temporal pattern of Ca2+ pulses. We speculate that such a mechanism may allow arrays of CaM kinase II molecules in postsynaptic densities to act as synaptic frequency detectors involved in setting the direction and level of synaptic modification. 相似文献