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.

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.

Effect of reduced myristoylated alanine-rich C kinase substrate expression on hippocampal mossy fiber development and spatial learning in mutant mice: Transgenic rescue and interactions with gene background

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent protein kinase C (PKC) substrate in brain that is expressed highly in hippocampal granule cells and their axons, the mossy fibers. Here, we examined hippocampal infrapyramidal mossy fiber (IP-MF) limb length and spatial learning in heterozygous Macs mutant mice that exhibit an ≈50% reduction in MARCKS expression relative to wild-type controls. On a 129B6(N3) background, the Macs mutation produced IP-MF hyperplasia, a significant increase in hippocampal PKCɛ expression, and proficient spatial learning relative to wild-type controls. However, wild-type 129B6(N3) mice exhibited phenotypic characteristics resembling inbred 129Sv mice, including IP-MF hypoplasia relative to inbred C57BL/6J mice and impaired spatial-reversal learning, suggesting a significant contribution of 129Sv background genes to wild-type and possibly mutant phenotypes. Indeed, when these mice were backcrossed with inbred C57BL/6J mice for nine generations to reduce 129Sv background genes, the Macs mutation did not effect IP-MF length or hippocampal PKCɛ expression and impaired spatial learning relative to wild-type controls, which now showed proficient spatial learning. Moreover, in a different strain (B6SJL(N1), the Macs mutation also produced a significant impairment in spatial learning that was reversed by transgenic expression of MARCKS. Collectively, these data indicate that the heterozygous Macs mutation modifies the expression of linked 129Sv gene(s), affecting hippocampal mossy fiber development and spatial learning performance, and that MARCKS plays a significant role in spatial learning processes.

Identification of a new class of protein kinases represented by eukaryotic elongation factor-2 kinase

The several hundred members of the eukaryotic protein kinase superfamily characterized to date share a similar catalytic domain structure, consisting of 12 conserved subdomains. Here we report the existence and wide occurrence in eukaryotes of a protein kinase with a completely different structure. We cloned and sequenced the human, mouse, rat, and Caenorhabditis elegans eukaryotic elongation factor-2 kinase (eEF-2 kinase) and found that with the exception of the ATP-binding site, they do not contain any sequence motifs characteristic of the eukaryotic protein kinase superfamily. Comparison of different eEF-2 kinase sequences reveals a highly conserved region of ≈200 amino acids which was found to be homologous to the catalytic domain of the recently described myosin heavy chain kinase A (MHCK A) from Dictyostelium. This suggests that eEF-2 kinase and MHCK A are members of a new class of protein kinases with a novel catalytic domain structure.

TAK, an HIV Tat-associated kinase, is a member of the cyclin-dependent family of protein kinases and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines

We have previously identified a cellular protein kinase activity termed TAK that specifically associates with the HIV types 1 and 2 Tat proteins. TAK hyperphosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase II in vitro in a manner believed to activate transcription [Herrmann, C. H. & Rice, A. P. (1995) J. Virol. 69, 1612–1620]. We show here that the catalytic subunit of TAK is a known human kinase previously named PITALRE, which is a member of the cyclin-dependent family of proteins. We also show that TAK activity is elevated upon activation of peripheral blood mononuclear cells and peripheral blood lymphocytes and upon differentiation of U1 and U937 promonocytic cell lines to macrophages. Therefore, in HIV-infected individuals TAK may be induced in T cells following activation and in macrophages following differentiation, thus contributing to high levels of viral transcription and the escape from latency of transcriptionally silent proviruses.

Eukaryotic phytochromes: Light-regulated serine/threonine protein kinases with histidine kinase ancestry

The discovery of cyanobacterial phytochrome histidine kinases, together with the evidence that phytochromes from higher plants display protein kinase activity, bind ATP analogs, and possess C-terminal domains similar to bacterial histidine kinases, has fueled the controversial hypothesis that the eukaryotic phytochrome family of photoreceptors are light-regulated enzymes. Here we demonstrate that purified recombinant phytochromes from a higher plant and a green alga exhibit serine/threonine kinase activity similar to that of phytochrome isolated from dark grown seedlings. Phosphorylation of recombinant oat phytochrome is a light- and chromophore-regulated intramolecular process. Based on comparative protein sequence alignments and biochemical cross-talk experiments with the response regulator substrate of the cyanobacterial phytochrome Cph1, we propose that eukaryotic phytochromes are histidine kinase paralogs with serine/threonine specificity whose enzymatic activity diverged from that of a prokaryotic ancestor after duplication of the transmitter module.

Phosphatidylinositol 3-Kinase-mediated Endocytosis of Renal Na+,K+-ATPase α Subunit in Response to Dopamine

Dopamine (DA) inhibition of Na+,K+-ATPase in proximal tubule cells is associated with increased endocytosis of its α and β subunits into early and late endosomes via a clathrin vesicle-dependent pathway. In this report we evaluated intracellular signals that could trigger this mechanism, specifically the role of phosphatidylinositol 3-kinase (PI 3-K), the activation of which initiates vesicular trafficking and targeting of proteins to specific cell compartments. DA stimulated PI 3-K activity in a time- and dose-dependent manner, and this effect was markedly blunted by wortmannin and LY 294002. Endocytosis of the Na+,K+-ATPase α subunit in response to DA was also inhibited in dose-dependent manner by wortmannin and LY 294002. Activation of PI 3-K generally occurs by association with tyrosine kinase receptors. However, in this study immunoprecipitation with a phosphotyrosine antibody did not reveal PI 3-K activity. DA-stimulated endocytosis of Na+,K+-ATPase α subunits required protein kinase C, and the ability of DA to stimulate PI 3-K was blocked by specific protein kinase C inhibitors. Activation of PI 3-K is mediated via the D1 receptor subtype and the sequential activation of phospholipase A2, arachidonic acid, and protein kinase C. The results indicate a key role for activation of PI 3-K in the endocytic sequence that leads to internalization of Na+,K+-ATPase α subunits in response to DA, and suggest a mechanism for the participation of protein kinase C in this process.

Yeast Dam1p Is Required to Maintain Spindle Integrity during Mitosis and Interacts with the Mps1p Kinase

We have identified a mutant allele of the DAM1 gene in a screen for mutations that are lethal in combination with the mps1-1 mutation. MPS1 encodes an essential protein kinase that is required for duplication of the spindle pole body and for the spindle assembly checkpoint. Mutations in six different genes were found to be lethal in combination with mps1-1, of which only DAM1 was novel. The remaining genes encode a checkpoint protein, Bub1p, and four chaperone proteins, Sti1p, Hsc82p, Cdc37p, and Ydj1p. DAM1 is an essential gene that encodes a protein recently described as a member of a microtubule binding complex. We report here that cells harboring the dam1-1 mutation fail to maintain spindle integrity during anaphase at the restrictive temperature. Consistent with this phenotype, DAM1 displays genetic interactions with STU1, CIN8, and KAR3, genes encoding proteins involved in spindle function. We have observed that a Dam1p-Myc fusion protein expressed at endogenous levels and localized by immunofluorescence microscopy, appears to be evenly distributed along short mitotic spindles but is found at the spindle poles at later times in mitosis.

MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration

Exposure of eukaryotic cells to extracellular stimuli results in activation of mitogen-activated protein kinase (MAPK) cascades composed of MAPKs, MAPK kinases (MAP2Ks), and MAPK kinase kinases (MAP3Ks). Mammals possess a large number of MAP3Ks, many of which can activate the c-Jun N-terminal kinase (JNK) MAPK cascade when overexpressed, but whose biological function is poorly understood. We examined the function of the MAP3K MEK kinase 1 (MEKK1) in proinflammatory signaling. Using MEKK1-deficient embryonic stem cells prepared by gene targeting, we find that, in addition to its function in JNK activation by growth factors, MEKK1 is required for JNK activation by diverse proinflammatory stimuli, including tumor necrosis factor α, IL-1, double-stranded RNA, and lipopolysaccharide. MEKK1 is also essential for induction of embryonic stem cell migration by serum factors, but is not required for activation of other MAPKs or the IκB kinase signaling cascade.

Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors

Acting through a number of distinct pathways, many G protein-coupled receptors (GPCRs) activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. Recently, it has been shown that in some cases, clathrin-mediated endocytosis is required for GPCR activation of the ERK/MAPK cascade, whereas in others it is not. Accordingly, we compared ERK activation mediated by a GPCR that does not undergo agonist-stimulated endocytosis, the α2A adrenergic receptor (α2A AR), with ERK activation mediated by the β2 adrenergic receptor (β2 AR), which is endocytosed. Surprisingly, we found that in COS-7 cells, ERK activation by the α2A AR, like that mediated by both the β2 AR and the epidermal growth factor receptor (EGFR), is sensitive to mechanistically distinct inhibitors of clathrin-mediated endocytosis, including monodansylcadaverine, a mutant dynamin I, and a mutant β-arrestin 1. Moreover, we determined that, as has been shown for many other GPCRs, both α2A and β2 AR-mediated ERK activation involves transactivation of the EGFR. Using confocal immunofluorescence microscopy, we found that stimulation of the β2 AR, the α2A AR, or the EGFR each results in internalization of a green fluorescent protein-tagged EGFR. Although β2 AR stimulation leads to redistribution of both the β2 AR and EGFR, activation of the α2A AR leads to redistribution of the EGFR but the α2A AR remains on the plasma membrane. These findings separate GPCR endocytosis from the requirement for clathrin-mediated endocytosis in EGFR transactivation-mediated ERK activation and suggest that it is the receptor tyrosine kinase or another downstream effector that must engage the endocytic machinery.

Stimulation of phosphatidylinositol 3-kinase by fibroblast growth factor receptors is mediated by coordinated recruitment of multiple docking proteins

The docking protein FRS2 is a major downstream effector that links fibroblast growth factor (FGF) and nerve growth factor receptors with the Ras/mitogen-activated protein kinase signaling cascade. In this report, we demonstrate that FRS2 also plays a pivotal role in FGF-induced recruitment and activation of phosphatidylinositol 3-kinase (PI3-kinase). We demonstrate that tyrosine phosphorylation of FRS2α leads to Grb2-mediated complex formation with the docking protein Gab1 and its tyrosine phosphorylation, resulting in the recruitment and activation of PI3-kinase. Furthermore, Grb2 bound to tyrosine-phosphorylated FRS2 through its SH2 domain interacts primarily via its carboxyl-terminal SH3 domain with a proline-rich region in Gab1 and via its amino-terminal SH3 domain with the nucleotide exchange factor Sos1. Assembly of FRS2α:Grb2:Gab1 complex induced by FGF stimulation results in activation of PI3-kinase and downstream effector proteins such as the S/T kinase Akt, whose cellular localization and activity are regulated by products of PI3-kinase. These experiments reveal a unique mechanism for generation of signal diversity by growth factor-induced coordinated assembly of a multidocking protein complex that can activate the Ras/mitogen-activated protein kinase cascade to induce cell proliferation and differentiation, and PI3-kinase to activate a mediator of a cell survival pathway.

The cytokine-activated tyrosine kinase JAK2 activates Raf-1 in a p21ras-dependent manner.

JAK2, a member of the Janus kinase superfamily was found to interact functionally with Raf-1, a central component of the ras/mitogen-activated protein kinase signal transduction pathway. Interferon-gamma and several other cytokines that are known to activate JAK2 kinase were also found to stimulate Raf-1 kinase activity toward MEK-1 in mammalian cells. In the baculovirus coexpression system, Raf-1 was activated by JAK2 in the presence of p21ras. Under these conditions, a ternary complex of p21ras, JAK2, and Raf-1 was observed. In contrast, in the absence of p21ras, coexpression of JAK2 and Raf-1 resulted in an overall decrease in the Raf-1 kinase activity. In addition, JAK2 phosphorylated Raf-1 at sites different from those phosphorylated by pp60v-src. In mammalian cells treated with either erythropoietin or interferon-gamma, a small fraction of Raf-1 coimmunoprecipitated with JAK2 in lysates of cells in which JAK2 was activated as judged by its state of tyrosine phosphorylation. Taken together, these data suggest that JAK2 and p21ras cooperate to activate Raf-1.

Cloning of rat MEK kinase 1 cDNA reveals an endogenous membrane-associated 195-kDa protein with a large regulatory domain.

The coding sequence of rat MEK kinase 1 (MEKK1) has been determined from multiple, independent cDNA clones. The cDNA is full-length based on the presence of stop codons in all three reading frames of the 5' untranslated region. Probes from the 5' and the 3' coding sequences both hybridize to a 7-kb mRNA. The open reading frame is 4.5 kb and predicts a protein with molecular mass of 161,225 Da, which is twice the size of the previously published MEKK1 sequence and reveals 801 amino acids of novel coding sequence. The novel sequence contains two putative pH domains, two proline-rich regions, and a cysteine-rich region. Antisera to peptides derived from this new sequence recognize an endogenous protein in human and rodent cells of 195 kDa, consistent with the size of the expressed rat MEKK1 clone. Endogenous and recombinant rat MEKK1 are enriched in membranes; little of either is found in soluble fractions. Expression of recombinant rat MEKK1 leads to activation of three mitogen-activated protein kinase modules in the order c-Jun N-terminal kinase/stress-activated protein kinase > p38 mitogen-activated protein kinase = extracellular signal-regulated kinase 2.

Nuclear export of late HIV-1 mRNAs occurs via a cellular protein export pathway.

The Rev protein of HIV-1 is essential for the nuclear export of incompletely spliced viral mRNAs. This action depends on the mutationally defined Rev activation domain, which both binds the nucleoporin-like human cellular cofactor Rab/hRIP and also functions as a nuclear export signal. Protein kinase inhibitor alpha (PKI) also contains a potent nuclear export signal. However, PKI plays no role in nuclear RNA export and instead induces the nuclear export of a specific protein target, the catalytic subunit of cAMP-dependent protein kinase. Here, it is demonstrated that the nuclear export signal of PKI not only binds the Rab/hRIP cofactor specifically but also can effectively substitute for the Rev activation domain in mediating the nuclear export of HIV-1 mRNAs. We conclude that HIV-1 Rev and PKI act through an identical nuclear export pathway and that Rev, rather than using a dedicated RNA export pathway, is instead acting as an adaptor that allows viral mRNAs to access a cellular protein export pathway.

The Bcr-Abl leukemia oncogene activates Jun kinase and requires Jun for transformation.

The leukemogenic tyrosine kinase fusion protein Bcr-Abl activates a Ras-dependent pathway required for transformation. To examine subsequent signal transduction events we measured the effect of Bcr-Abl on two mitogen-activated protein kinase (MAPK) cascades--the extracellular signal-regulated kinase (ERK) pathway and the Jun N-terminal kinase (JNK) pathway. We find that Bcr-Abl primarily activates JNK in fibroblasts and hematopoietic cells. Bcr-Abl enhances JNK function as measured by transcription from Jun responsive promoters and requires Ras, MEK kinase (MAPK/ERK kinase kinase), and JNK to do so. Dominant-negative mutants of c-Jun, which inhibit the endpoint of the JNK pathway, impair Bcr-Abl transforming activity. These findings implicate the JNK pathway in transformation by a human leukemia oncogene.