Phosphorylation of serine 4642 in the C-terminus of plectin by MNK2 and PKA modulates its interaction with intermediate filaments

Plectin is a versatile cytolinker of the plakin family conferring cell resilience to mechanical stress in stratified epithelia and muscles. It acts as a critical organizer of the cytoskeletal system by tethering various intermediate filament (IF) networks through its C-terminal IF-binding domain (IFBD). Mutations affecting the IFBD cause devastating human diseases. Here, we show that serine 4642, which is located in the extreme C-terminus of plectin, is phosphorylated in different cell lines. Phosphorylation of S4642 decreased the ability of plectin IFBD to associate with various IFs, as assessed by immunofluorescence microscopy and cell fractionation studies, as well as in yeast two-hybrid assays. Plectin phosphorylated at S4642 was reduced at sites of IF network anchorage along cell-substrate contacts in both skin and cultured keratinocytes. Treatment of SK-MEL-2 and HeLa cells with okadaic acid increased plectin S4642 phosphorylation, suggesting that protein phosphatase 2A dephosphorylates this residue. Moreover, plectin S4642 phosphorylation was enhanced after cell treatment with EGF, phorbol ester, sorbitol and 8-bromo-cyclic AMP, as well as during wound healing and protease-mediated cell detachment. Using selective protein kinase inhibitors, we identified two different kinases that modulate the phosphorylation of plectin S4642 in HeLa cells: MNK2, which is downstream of the ERK1/2-dependent MAPK cascade, and PKA. Our study indicates that phosphorylation of S4642 has an important regulatory role in the interaction of plectin with IFs and identifies a novel link between MNK2 and the cytoskeleton.

Activation of Src kinase Lyn by the Kaposi sarcoma-associated herpesvirus K1 protein: implications for lymphomagenesis.

The K1 gene of Kaposi sarcoma-associated herpesvirus (KSHV) encodes a transmembrane glycoprotein bearing a functional immunoreceptor tyrosine-based activation motif (ITAM). Previously, we reported that the K1 protein induced plasmablastic lymphomas in K1 transgenic mice, and that these lymphomas showed enhanced Lyn kinase activity. Here, we report that systemic administration of the nuclear factor kappa B (NF-kappaB) inhibitor Bay 11-7085 or an anti-vascular endothelial growth factor (VEGF) antibody significantly reduced K1 lymphoma growth in nude mice. Furthermore, in KVL-1 cells, a cell line derived from a K1 lymphoma, inhibition of Lyn kinase activity by the Src kinase inhibitor PP2 decreased VEGF induction, NF-kappaB activity, and the cell proliferation index by 50% to 75%. In contrast, human B-cell lymphoma BJAB cells expressing K1, but not the ITAM sequence-deleted mutant K1, showed a marked increase in Lyn kinase activity with concomitant VEGF induction and NF-kappaB activation, indicating that ITAM sequences were required for the Lyn kinase-mediated activation of these factors. Our results suggested that K1-mediated constitutive Lyn kinase activation in K1 lymphoma cells is crucial for the production of VEGF and NF-kappaB activation, both strongly implicated in the development of KSHV-induced lymphoproliferative disorders.

Characterization of the potential role for RNA-binding protein FUS in DNA damage response: A quantitative proteomic approach

FUS/TLS (fused in sarcoma/translocated in liposarcoma) is a ubiquitously expressed protein of the hnRNP family, that has been discovered as fused to transcription factors in several human sarcomas and found in protein aggregates in neurons of patients with an inherited form of Amyotrophic Lateral Sclerosis [Vance C. et al., 2009]. FUS is a 53 kDa nuclear protein that contains structural domains, such as a RNA Recognition Motif (RRM) and a zinc finger motif, that give to FUS the ability to bind to both RNA and DNA sequences. It has been implicated in a variety of cellular processes, such as pre-mRNA splicing, miRNA processing, gene expression control and transcriptional regulation [Fiesel FC. and Kahle PJ., 2011]. Moreover, some evidences link FUS to genome stability control and DNA damage response: mice lacking FUS are hypersensitive to ionizing radiation (IR) and show high levels of chromosome instability and, in response to double-strand breaks, FUS is phosphorylated by the protein kinase ATM [Kuroda M. et al., 2000; Hicks GG. et al., 2000; Gardiner M. et al., 2008]. Furthermore, preliminary results of mass spectrometric identification of FUS interacting proteins in HEK293 cells, expressing a recombinant flag-tagged FUS protein, highlighted the interactions with proteins involved in DNA damage response, such as DNA-PK, XRCC-5/-6, and ERCC-6, raising the possibilities that FUS is involved in this pathway, even though its role still needs to be clarified. This study aims to investigate the biological roles of FUS in human cells and in particular the putative role in DNA damage response through the characterization of the proteomic profile of the neuroblastoma cell line SH-SY5Y upon FUS inducible depletion, by a quantitative proteomic approach. The SH-SY5Y cell line that will be used in this study expresses, in presence of tetracycline, a shRNA that targets FUS mRNA, leading to FUS protein depletion (SH-SY5Y FUS iKD cells). To quantify changes in proteins expression levels a SILAC strategy (Stable Isotope Labeling by Amino acids in Cell culture) will be conducted on SH-SY5Y FUS iKD cells and a control SH-SY5Y cell line (that expresses a mock shRNA) and the relative changes in proteins levels will be evaluated after five and seven days upon FUS depletion, by nanoliquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS) and bioinformatics analysis. Preliminary experiments demonstrated that the SH-SY5Y FUS iKD cells, when subjected to genotoxic stress (high dose of IR), upon inducible depletion of FUS, showed a increased phosphorylation of gH2AX with respect to control cells, suggesting an higher activation of the DNA damage response.

RNA interference screening identifies a novel role for PCTK1/CDK16 in medulloblastoma with c-Myc amplification.

Medulloblastoma (MB) is the most common malignant brain tumor in children and is associated with a poor outcome. cMYC amplification characterizes a subgroup of MB with very poor prognosis. However, there exist so far no targeted therapies for the subgroup of MB with cMYC amplification. Here we used kinome-wide RNA interference screening to identify novel kinases that may be targeted to inhibit the proliferation of c-Myc-overexpressing MB. The RNAi screen identified a set of 5 genes that could be targeted to selectively impair the proliferation of c-Myc-overexpressing MB cell lines: AKAP12 (A-kinase anchor protein), CSNK1α1 (casein kinase 1, alpha 1), EPHA7 (EPH receptor A7) and PCTK1 (PCTAIRE protein kinase 1). When using RNAi and a pharmacological inhibitor selective for PCTK1, we could show that this kinase plays a crucial role in the proliferation of MB cell lines and the activation of the mammalian target of rapamycin (mTOR) pathway. In addition, pharmacological PCTK1 inhibition reduced the expression levels of c-Myc. Finally, targeting PCTK1 selectively impaired the tumor growth of c-Myc-overexpressing MB cells in vivo. Together our data uncover a novel and crucial role for PCTK1 in the proliferation and survival of MB characterized by cMYC amplification.

CHARACTERIZATION OF SERINE KINASE ACTIVITY ASSOCIATED WITH MOLONEY MURINE SARCOMA VIRUS V-MOS GENE PRODUCTS

Various Moloney murine sarcoma virus (Mo-MuSV) isolates contain a cellular sequence, termed mos, which is responsible for the transforming ability of Mo-MuSV. A serine kinase activity has been found to be associated with mos gene products of several isolates of Mo-MuSV. A mutant of Mo-MuSV strain 124 (designated MuSV ts110) is temperature-sensitive (ts) for transformation and encodes two proteins, P85('gag-mos) (an 85,000 M(,r) protein encoded by the gag and mos genes) and P58('gag), at the permissive temperature (28(DEGREES)C). At the nonpermissive temperature (39(DEGREES)C), only P58('gag) is found in MuSV ts110-infected NRK cells (6m2 cells). Both P85('gag-mos) and P58('gag) were phosphorylated when anti-gag immune complexes containing these proteins were incubated at 22(DEGREES)C with (lamda)-('32)P -ATP and MnCl(,2). The kinase detected in anti-gag complexes from 6m2 cells at permissive temperature was associated with P85('gag-mos) since immune complexes from 39(DEGREES)C 6m2 cells, which lack P85('gag-mos), produced no phosphorylated P58('gag) molecules. In addition, an anti-mos complex (anti-mos 37-55 complexes) allowed in vitro phosphorylation of P85('gag-mos) in the absence of P58('gag). No kinase activity was detectable with other gag gene products (e.g., Mo-MuSV-124 P62('gag)), suggesting that the P85('gag-mos) kinase activity was present within the mos portion of the protein. The P85('gag-mos) kinase activity was very thermolabile upon shifting 6m2 cells from permissive to nonpermissive temperatures (t(, 1/2) for inactivation = 5 min). In contrast, a spontaneous revertant of MuSV ts110 encodes a larger gag-mos protein (termed P100('gag-mos)) which contained a kinase activity stable to 39(DEGREES)C. Using the optimal conditions developed for the P85('gag-mos) kinase, Mo-MuSV-encoded p37('mos) was also found to be associated with a serine kinase activity. Phosphorylation of p37('mos) and a 43 Kd protein (super-phosphorylated p37('mos)) occurred in anti-mos(37-55) complexes from Mo-MuSV-124 acutely-infected NIH 3T3 cells, but neither in mos 37-55 peptide-blocked anti-mos(37-55) complexes nor in immune complexes from uninfected NIH 3T3 cells. Antibodies directed against the C-terminus of v-mos were found to inhibit the in vitro phosphorylation of p37('mos), suggesting that the extreme C-terminal sequence of v-mos may be important for an intrinsic kinase activity. This inhibitory action by antibodies to the C-terminus of p37('mos), when considered with all the other data reported here, provides convincing evidence that the v-mos gene encodes a serine protein kinase activity. ^

Biochemical and genetic analysis of two protein kinases JNKK2 and MEKK3

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryotic signaling modules consisting of a MAPK, a MAPKK and a MAP3K. MAPK cascades are involved in many cellular responses including proliferation, differentiation, apoptosis, stress and immune responses. ^ The first part of this thesis describes the cloning and biochemical analysis of JNKK2, a member of MAPKK gene family. Our results demonstrate that JNKK2 is a specific JNK activator and activates the JNK-dependent signal transduction pathway in vivo by inducing c-Jun and ATF2-mediated gene expression. We also found that JNKK2 is specifically activated by a MAP3K MEKK2 through formation of MEKK2-JNKK2-JNK1 triple complex module. JNKK2 is likely to mediate specific upstream signals to activate JNK cascade. ^ The second part of this thesis describes biochemical and gene disruption analysis of MEKK3, a member of MAP3K gene family. We showed that overexpression of MEKK3 strongly activates both JNK and p38 MAPKs but only weakly activates ERK. MEKK−/− embryos die at about embryonic day (E) 11. MEKK3−/− embryos displayed defects in blood vessel development in the yolk sacs, and in the myocardium and endocardium development at E9.5. The angiogenesis in the head, intersomitic region and placenta was also abnormal. These results demonstrate that MEKK3, a member of MAP3K MEKK/STE11 subgene family, is essential for early embryonic cardiovascular development. Furthermore, it was found that disruption of MEKK3 did not alter the expression of vascular endothelial growth factor-1 (VEGF-1), angiopoietin-1, -2 and their respective receptors Flt-1, Flk-1, Tie-1, Tie-2. Finally, MEKK3 was shown to activate myocyte-specific enhancer factor 2C (MEF2C), a crucial transcription factor for early embryonic cardiovascular development through the p38 MAPK cascade, suggesting that MEF2C is one of the key targets of the MEEKK3 signaling pathway during early embryonic cardiovascular development. ^

p21 activated kinase 5 links multiple GTPase signaling pathways at mitochondria

The p21-activated kinase 5 (PAK5) is a serine/threonine protein kinase associated with the group 2 subfamily of PAKs. Although our understanding about PAK5 is very limited, it is receiving increasing interest due to its tissue specific expression pattern and important signaling properties. PAK5 is highly expressed in brain. Its overexpression induces neurite outgrowth in neuroblastoma cells and promotes survival in fibroblasts. ^ The serine/threonine protein kinase Raf-1 is an essential mediator of Ras-dependent signaling that controls the ERK/MAPK pathway. In contrast to PAK5, Raf-1 has been the subject of intensive investigation. However due to the complexity of its activation mechanism, the biological inputs controlling Raf-1 activation are not fully understood. ^ PAKs 1-3 are the known kinases responsible for phosphorylation of Raf-1 on serine 338, which is a crucial phosphorylation site for Raf-1 activation. However, dominant negative versions of these kinases do not block EGF-induced Raf-1 activation, indicating that other kinases may regulate the phosphorylation of Raf-1 on serine 338. ^ This thesis work was initiated to test whether the group 2 PAKs 4, 5 and 6 are responsible for EGF-induced Raf-1 activation. We found that PAK5, and to a lesser extent PAK4, can activate Raf-1 in cells. Our studies thereafter focused on PAK5. With the progress of our study we found that PAK5 does not significantly stimulate serine 338 phosphorylation of Triton X-100 soluble Raf-1. PAK5, however, constitutively and specifically associates with Raf-1 and targets it to a Triton X-100 insoluble, mitochondrial compartment, where PAK5 phosphorylates serine 338 of Raf-1. We further demonstrated that endogenous PAK5 and Raf-1 colocalize in Hela cells at the mitochondrial outer membrane. In addition, we found that the mitochondria-targeting of PAK5 is determined by its C-terminal kinase domain plus the upstream proximal region, and facilitated by the N-terminal p21 binding domain. We also demonstrated that Rho GTPases Cdc42 and RhoD associate with and regulate the subcellular localization of PAK5. Taken together, this work suggests that the mitochondria-targeting of PAK5 may link Ras and Rho GTPase-mediated signaling pathways, and sheds light on aspects of PAK5 signaling that may be important for regulating neuronal homeostasis. ^

Oncogenic activation of c-Abl tyrosine kinase in non-small cell lung cancer: FUS1 tumor suppressor down-regulates c-Abl tyrosine kinase

The FUS1 tumor suppressor gene (TSG) has been found to be deficient in many human non-small cell lung cancer (NSCLC) tissue samples and cell lines (1,2,3). Studies have shown potent anti-tumor activity of FUS1 in animal models where FUS1 was delivered through a liposomal vector (4) and the use of FUS1 as a therapeutic agent is currently being studied in clinical human trials (5). Currently, the mechanisms of FUS1 activity are being investigated and my studies have shown that c-Abl tyrosine kinase is inhibited by the FUS1 TSG.^ Considering that many NSCLC cell lines are FUS1 deficient, my studies further identified that FUS1 deficient NSCLC cells have an activated c-Abl tyrosine kinase. C-Abl is a known proto-oncogene and while c-Abl kinase is tightly regulated in normal cells, constitutively active Abl kinase is known to contribute to the oncogenic phenotype in some types of hematopoietic cancers. My studies show that the active c-Abl kinase contributes to the oncogenicity of NSCLC cells, particularly in tumors that are deficient in FUS1, and that c-Abl may prove to be a viable target in NSCLC therapy.^ Current studies have shown that growth factor receptors play a role in NSCLC. Over-expression of the epidermal growth factor receptor (EGFR) plays a significant role in aggressiveness of NSCLC. Current late stage treatments include EFGR tyrosine kinase inhibitors or EGFR antibodies. Platelet-derived growth factor receptor (PDGFR) also has been shown to play a role in NSCLC. Of note, both growth factor receptors are known upstream activators of c-Abl kinase. My studies indicate that growth factor receptor simulation along deficiency in FUS1 expression contributes to the activation of c-Abl kinase in NSCLC cells. ^

Regulation of v-Mos kinase activity by phosphorylation

In this thesis, I investigated the effect of cylic AMP-dependent protein kinase (PKA) on v-Mos kinase activity. Increase in PKA activity in vivo brought about either by forskolin treatment or by overexpression of the PKA catalytic subunit resulted in a significant inhibition of v-Mos kinase activity. The purified PKA catalytic subunit was able to phosphorylate recombinant p37$\rm\sp{v-mos}$ in vitro, suggesting that the mechanism of in vivo inhibition of v-Mos kinase involves direct phosphorylation by PKA. Ser-263 was identified as a residue that is normally phosphorylated at a very low level but whose phosphorylation is dramatically increased upon forskolin treatment. Consistent with the inhibitory role of Ser-263 phosphorylation, the Ala-263 mutant of v-Mos was not inhibited by forskolin treatment. Based on our results, we propose that the known inhibitory role of PKA in the initiation of oocyte maturation could be explained at least in part by its inhibition of Mos kinase.^ Combining tryptic phosphopeptide two-dimensional mapping analysis and in vitro mutagenesis studies, I identified Ser-56 as the major in vivo phosphorylation site on v-Mos. I studied the interrelationship between Ser-34 and Ser-56 phosphorylation in regulating v-Mos function. After site-directed mutagenesis to substitute serine residues with alanine or glutamic acid in different combinations to mimick unphosphorylated and phosphorylated serines respectively, various v-Mos mutants were expressed in COS-1 cells. As expected, Ala-34 mutant of v-Mos had very low (less 5% of wild type) kinase activity. The Ala-56 mutant had kinase activity 50% that of wild type. Surprisingly, the Ala-34 Ala-56 double mutant and the Ala-56 mutant exhibited identical kinase activity. On the other hand, Ala-34 Glu-56 double mutant had reduced kinase activity comparable to Ala-34 mutant. These results suggest that the phosphorylation at Ser-56 may serve to inhibit the activation of newly synthesized Mos protein. As predicted from Xenopus c-Mos studies, Glu-34 mutant of v-Mos was highly active (125% that of wild type). Interestingly, consistant with the model involving an inhibitory role of Ser-56 phosphorylation, the Glu-34 Glu-56 double mutant was totally inactive as a kinase. Moreover in my experiments, there was a perfect correlation between the level of v-Mos kinase activity of various mutants and their transforming activity. The latter is dependent upon MEK1 phosphorylation/ activation in v-mos transformed cells. Residues corresponding to both v-Mos Ser-34 and Ser-56 are evolutionarily conserved in c-Mos. Therefore, the cytostatic factor function of c-Mos may be regulated in the same manner as v-Mos kinase activity.^ It has been known that v-mos transforms cells by affecting G1 phase progression of the cell cycle. Here I showed that mos induces cyclin D1 expression in mos transformed NIH 3T3 cells and NRK 6m2 cells, and this induced level was found to be unaffected by serum starvation. Consequently, cyclin D1-Cdk4 and cyclin E-Cdk2 activities increase, and retinoblastoma protein is hyperphosphorylated. Based on studies from several laboratories, these findings suggest that increased amount of cyclin D1-Cdk4 complexes ties up the limited amount of cyclin E-Cdk2 inhibitors (e.g. p27), causing the activation of cyclin E-Cdk2. My results indicate that activation of key cell cycle regulators of G1 phase may be important for cellular transformation by mos. (Abstract shortened by UMI.) ^

Role and regulation of c-KIT gene expression in tumor growth and metastasis of human melanoma

The purpose of this study was to investigate the role of the c-KIT receptor in the progression of human melanoma and the mechanism(s) for the regulation of c-KIT gene expression in human melanoma.^ The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase (VGP) (metastatic phenotype) are not well-defined. Expression of the tyrosine-kinase receptor c-KIT progressively decreases during local tumor growth and invasion of human melanomas. To provide direct evidence that the metastasis of human melanoma is associated with the loss of c-KIT expression, highly metastatic A375SM cells, which express very low or undetectable levels of c-KIT, were tranduced with the human c-KIT gene. We demonstrated that enforced c-KIT expression in highly metastatic human melanoma cells significantly suppressed their tumorigenicity and metastatic propensity in nude mice. In addition, we showed that the ligand for c-KIT, SCF, induces apoptosis in human melanoma cells expressing c-KIT under both in vitro and in vivo conditions. These results suggest that loss of c-KIT receptor may allow malignant melanoma cells to escape SCF/c-KIT-mediated apoptosis, thus contributing to tumor growth and eventually metastasis.^ Furthermore, we investigated the possible mechanism(s) for the down-regulation of c-KIT gene expression in malignant melanoma. Sequence analysis of the c-KIT promoter indicated that this promoter contains several consensus binding-site sequences including three putative AP2 and two Myb sites. Although Myb was shown to be associated with c-KIT expression in human hemotopoietic cells, we found no correlation between c-KIT expression and Myb expression in human melanoma cell lines. In contrast, we showed that c-KIT expression directly correlates with expression of AP2 in human melanoma cells. We found that highly metastatic cells do not express the transcription factor AP2. Expression of AP2 in A375SM cells (c-KIT-negative and AP2-negative) was enough to restore luciferase activity driven by the c-KIT promoter in a dose-dependent manner. On the other hand, co-expression of the dominant-negative form of AP2 (AP2B) in Mel-501 cells (c-KIT-positive and AP2-positive) resulted in two-fold reduction in luciferase activity. Electrophoretic mobility shift assays revealed that the c-KIT promoter contains functional AP2 binding sites which could associate with AP2 protein. Endogenous c-KIT gene expression levels were elevated in AP2 stably-transfected human melanoma A375SM cells. Expression of exogenous AP2 in A375SM cells inhibited their tumorigenicity and metastatic potential in nude mice. The c-KIT ligand, SCF, also induced apoptosis in the AP2 stably-transfected A375SM cells. The identification of AP2 as an important regulator for c-KIT expression suggests that AP2 may have tumor growth and metastasis inhibitory properties, possibly mediated through c-KIT/SCF effects on apoptosis of human melanoma cells. Since AP2 binding sites were found in the promoters of other genes involved in the progression of human melanoma, such as MMP2 (72 kDa collagenase), MCAM/MUC18 and P21/WAF-1, our findings suggest that loss of AP2 expression might be a crucial event in the development of malignant melanoma. ^

Disruption of cellular translational control by a viral truncated eukaryotic translation initiation factor 2α kinase homolog

Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) is a common cellular mechanism to limit protein synthesis in stress conditions. Baculovirus PK2, which resembles the C-terminal half of a protein kinase domain, was found to inhibit both human and yeast eIF2α kinases. Insect cells infected with wild-type, but not pk2-deleted, baculovirus exhibited reduced eIF2α phosphorylation and increased translational activity. The negative regulatory effect of human protein kinase RNA-regulated (PKR), an eIF2α kinase, on virus production was counteracted by PK2, indicating that baculoviruses have evolved a unique strategy for disrupting a host stress response. PK2 was found in complex with PKR and blocked kinase autophosphorylation in vivo, suggesting a mechanism of kinase inhibition mediated by interaction between truncated and intact kinase domains.

Expressed protein ligation: A general method for protein engineering

A protein semisynthesis method—expressed protein ligation—is described that involves the chemoselective addition of a peptide to a recombinant protein. This method was used to ligate a phosphotyrosine peptide to the C terminus of the protein tyrosine kinase C-terminal Src kinase (Csk). By intercepting a thioester generated in the recombinant protein with an N-terminal cysteine containing synthetic peptide, near quantitative chemical ligation of the peptide to the protein was achieved. The semisynthetic tail-phosphorylated Csk showed evidence of an intramolecular phosphotyrosine-Src homology 2 interaction and an unexpected increase in catalytic phosphoryl transfer efficiency toward a physiologically relevant substrate compared with the non-tail-phosphorylated control. This work illustrates that expressed protein ligation is a simple and powerful new method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophysical probes into proteins of any size.

Mobilization of the A-kinase N-myristate through an isoform-specific intermolecular switch

Although the catalytic (C) subunit of cAMP-dependent protein kinase is N-myristylated, it is a soluble protein, and no physiological role has been identified for its myristyl moiety. To determine whether the interaction of the two regulatory (R) subunit isoforms (RI and RII) with the N-myristylated C subunit affects its ability to target membranes, the effect of N-myristylation and the RI and RII subunit isoforms on C subunit binding to phosphatidylcholine/phosphatidylserine liposomes was examined. Only the combination of N-myristylation and RII subunit interaction produced a dramatic increase in the rate of liposomal binding. To assess whether the RII subunit also increased the conformational flexibility of the C subunit N terminus, the effect of N-myristylation and the RI and RII subunits on the rotational freedom of the C subunit N terminus was measured. Specifically, fluorescein maleimide was conjugated to Cys-16 in the N-terminal domain of a K16C mutant of the C subunit, and the time-resolved emission anisotropy was determined. The interaction of the RII subunit, but not the RI subunit, significantly increased the backbone flexibility around the site of mutation and labeling, strongly suggesting that RII subunit binding to the myristylated C subunit induced a unique conformation of the C subunit that is associated with an increase in both the N-terminal flexibility and the exposure of the N-myristate. RII subunit thus appears to serve as an intermolecular switch that disrupts of the link between the N-terminal and core catalytic domains of the C subunit to expose the N-myristate and poise the holoenzyme for interaction with membranes.

Independent regulation of JNK/p38 mitogen-activated protein kinases by metabolic oxidative stress in the liver

The stress-activated protein kinases JNK and p38 mediate increased gene expression and are activated by environmental stresses and proinflammatory cytokines. Using an in vivo model in which oxidative stress is generated in the liver by intracellular metabolism, rapid protein–DNA complex formation on stress-activated AP-1 target genes was observed. Analysis of the induced binding complexes indicates that c-fos, c-jun, and ATF-2 were present, but also two additional jun family members, JunB and JunD. Activation of JNK precedes increased AP-1 DNA binding. Furthermore, JunB was shown to be a substrate for JNK, and phosphorylation requires the N-terminal activation domain. Unexpectedly, p38 activity was found to be constitutively active in the liver and was down-regulated through selective dephosphorylation following oxidative stress. One potential mechanism for p38 dephosphorylation is the rapid stress-induced activation of the phosphatase MKP-1, which has high affinity for phosphorylated p38 as a substrate. These data demonstrate that there are mechanisms for independent regulation of the JNK and p38 mitogen-activated protein kinase signal transduction pathways after metabolic oxidative stress in the liver.

Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans

The CST20 gene of Candida albicans was cloned by functional complementation of a deletion of the STE20 gene in Saccharomyces cerevisiae. CST20 encodes a homolog of the Ste20p/p65PAK family of protein kinases. Colonies of C. albicans cells deleted for CST20 revealed defects in the lateral formation of mycelia on synthetic solid “Spider” media. However, hyphal development was not impaired in some other media. A similar phenotype was caused by deletion of HST7, encoding a functional homolog of the S. cerevisiae Ste7p protein kinase. Overexpression of HST7 partially complemented the deletion of CST20. Cells deleted for CST20 were less virulent in a mouse model for systemic candidiasis. Our results suggest that more than one signaling pathway can trigger hyphal development in C. albicans, one of which has a protein kinase cascade that is analogous to the mating response pathway in S. cerevisiae and might have become adapted to the control of mycelial formation in asexual C. albicans.