The Polo-like Kinase Plx1 Is Required for Activation of the Phosphatase Cdc25C and Cyclin B-Cdc2 in Xenopus Oocytes

In the Xenopus oocyte system mitogen treatment triggers the G2/M transition by transiently inhibiting the cAMP-dependent protein kinase (PKA); subsequently, other signal transduction pathways are activated, including the mitogen-activated protein kinase (MAPK) and polo-like kinase pathways. To study the interactions between these pathways, we have utilized a cell-free oocyte extract that carries out the signaling events of oocyte maturation after addition of the heat-stable inhibitor of PKA, PKI. PKI stimulated the synthesis of Mos and activation of both the MAPK pathway and the Plx1/Cdc25C/cyclin B-Cdc2 pathway. Activation of the MAPK pathway alone by glutathione S-transferase (GST)-Mos did not lead to activation of Plx1 or cyclin B-Cdc2. Inhibition of the MAPK pathway in the extract by the MEK1 inhibitor U0126 delayed, but did not prevent, activation of the Plx1 pathway, and inhibition of Mos synthesis by cycloheximide had a similar effect, suggesting that MAPK activation is the only relevant function of Mos. Immunodepletion of Plx1 completely inhibited activation of Cdc25C and cyclin B-Cdc2 by PKI, indicating that Plx1 is necessary for Cdc25C activation. In extracts containing fully activated Plx1 and Cdc25C, inhibition of cyclin B-Cdc2 by p21Cip1 had no significant effect on either the phosphorylation of Cdc25C or the activity of Plx1. These results demonstrate that maintenance of Plx1 and Cdc25C activity during mitosis does not require cyclin B-Cdc2 activity.

The amphiphysin-like protein 1 (ALP1) interacts functionally with the cABL tyrosine kinase and may play a role in cytoskeletal regulation

cABL is a protooncogene, activated in a subset of human leukemias, whose protein product is a nonreceptor tyrosine kinase of unknown function. cABL has a complex structure that includes several domains and motifs found in proteins implicated in signal transduction pathways. An approach to elucidate cABL function is to identify proteins that interact directly with cABL and that may serve as regulators or effectors of its activity. To this end, a protein-interaction screen of a phage expression library was undertaken to identify proteins that interact with specific domains of cABL. An SH3-domain-containing protein has been identified that interacts with sequences in the cABL carboxyl terminus. The cDNA encoding ALP1 (amphiphysin-like protein 1) was isolated from a 16-day mouse embryo. ALP1 has high homology to BIN1, a recently cloned myc-interacting protein, and also shows significant homology to amphiphysin, a neuronal protein cloned from human and chicken. The amino terminus has homology to two yeast proteins, Rvs167 and Rvs161, which are involved in cell entry into stationary phase and cytoskeletal organization. ALP1 binds cABL in vitro and in vivo. Expression of ALP1 results in morphological transformation of NIH 3T3 fibroblasts in a cABL-dependent manner. The properties of ALP1 suggest that it may be involved in possible cytoskeletal functions of the cABL kinase. Additionally, these results provide further evidence for the importance of the cABL carboxyl terminus and its binding proteins in the regulation of cABL function.

The polo-box-dependent induction of ectopic septal structures by a mammalian polo kinase, Plk, in Saccharomyces cerevisiae

Members of the polo subfamily of protein kinases play pivotal roles in cell-cycle control and proliferation. In addition to a high degree of sequence similarity in the kinase domain, polo kinases contain a strikingly conserved motif termed “polo-box” in the noncatalytic C-terminal domain. We have previously shown that the mammalian polo-like kinase Plk is a functional homolog of Saccharomyces cerevisiae Cdc5. Here, we show that, in a polo-box- and kinase activity-dependent manner, ectopic expression of Plk in budding yeast can induce a class of cells with abnormally elongated buds. In addition to localization at spindle poles and cytokinetic neck filaments, Plk induces and localizes to ectopic septin ring structures within the elongated buds. In contrast, mutations in the polo-box abolish both localization to, and induction of, septal structures. Consistent with the polo-box-dependent subcellular localization, the C-terminal domain of Plk, but not its polo-box mutant, is sufficient for subcellular localization. Our data suggest that Plk may contribute a signal to initiate or promote cytokinetic event(s) and that an intact polo-box is required for regulation of these cellular processes.

Cofilin Phosphorylation by Protein Kinase Testicular Protein Kinase 1 and Its Role in Integrin-mediated Actin Reorganization and Focal Adhesion Formation

Testicular protein kinase 1 (TESK1) is a serine/threonine kinase with a structure composed of a kinase domain related to those of LIM-kinases and a unique C-terminal proline-rich domain. Like LIM-kinases, TESK1 phosphorylated cofilin specifically at Ser-3, both in vitro and in vivo. When expressed in HeLa cells, TESK1 stimulated the formation of actin stress fibers and focal adhesions. In contrast to LIM-kinases, the kinase activity of TESK1 was not enhanced by Rho-associated kinase (ROCK) or p21-activated kinase, indicating that TESK1 is not their downstream effector. Both the kinase activity of TESK1 and the level of cofilin phosphorylation increased by plating cells on fibronectin. Y-27632, a specific inhibitor of ROCK, inhibited LIM-kinase-induced cofilin phosphorylation but did not affect fibronectin-induced or TESK1-induced cofilin phosphorylation in HeLa cells. Expression of a kinase-negative TESK1 suppressed cofilin phosphorylation and formation of stress fibers and focal adhesions induced in cells plated on fibronectin. These results suggest that TESK1 functions downstream of integrins and plays a key role in integrin-mediated actin reorganization, presumably through phosphorylating and inactivating cofilin. We propose that TESK1 and LIM-kinases commonly phosphorylate cofilin but are regulated in different ways and play distinct roles in actin reorganization in living cells.

Expression of a β-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice

Heart failure is accompanied by severely impaired β-adrenergic receptor (βAR) function, which includes loss of βAR density and functional uncoupling of remaining receptors. An important mechanism for the rapid desensitization of βAR function is agonist-stimulated receptor phosphorylation by the βAR kinase (βARK1), an enzyme known to be elevated in failing human heart tissue. To investigate whether alterations in βAR function contribute to the development of myocardial failure, transgenic mice with cardiac-restricted overexpression of either a peptide inhibitor of βARK1 or the β2AR were mated into a genetic model of murine heart failure (MLP−/−). In vivo cardiac function was assessed by echocardiography and cardiac catheterization. Both MLP−/− and MLP−/−/β2AR mice had enlarged left ventricular (LV) chambers with significantly reduced fractional shortening and mean velocity of circumferential fiber shortening. In contrast, MLP−/−/βARKct mice had normal LV chamber size and function. Basal LV contractility in the MLP−/−/βARKct mice, as measured by LV dP/dtmax, was increased significantly compared with the MLP−/− mice but less than controls. Importantly, heightened βAR desensitization in the MLP−/− mice, measured in vivo (responsiveness to isoproterenol) and in vitro (isoproterenol-stimulated membrane adenylyl cyclase activity), was completely reversed with overexpression of the βARK1 inhibitor. We report here the striking finding that overexpression of this inhibitor prevents the development of cardiomyopathy in this murine model of heart failure. These findings implicate abnormal βAR-G protein coupling in the pathogenesis of the failing heart and point the way toward development of agents to inhibit βARK1 as a novel mode of therapy.

Essential role of β-adrenergic receptor kinase 1 in cardiac development and function

The β-adrenergic receptor kinase 1 (βARK1) is a member of the G protein-coupled receptor kinase (GRK) family that mediates the agonist-dependent phosphorylation and desensitization of G protein-coupled receptors. We have cloned and disrupted the βARK1 gene in mice by homologous recombination. No homozygote βARK1−/− embryos survive beyond gestational day 15.5. Prior to gestational day 15.5, βARK1−/− embryos display pronounced hypoplasia of the ventricular myocardium essentially identical to the “thin myocardium syndrome” observed upon gene inactivation of several transcription factors (RXRα, N-myc, TEF-1, WT-1). Lethality in βARK1−/− embryos is likely due to heart failure as they exhibit a >70% decrease in cardiac ejection fraction determined by direct in utero intravital microscopy. These results along with the virtual absence of endogenous GRK activity in βARK1−/− embryos demonstrate that βARK1 appears to be the predominant GRK in early embryogenesis and that it plays a fundamental role in cardiac development.

The Yck2 Yeast Casein Kinase 1 Isoform Shows Cell Cycle-specific Localization to Sites of Polarized Growth and Is Required for Proper Septin Organization

Casein kinase 1 protein kinases are ubiquitous and abundant Ser/Thr-specific protein kinases with activity on acidic substrates. In yeast, the products of the redundant YCK1 and YCK2 genes are together essential for cell viability. Mutants deficient for these proteins display defects in cellular morphogenesis, cytokinesis, and endocytosis. Yck1p and Yck2p are peripheral plasma membrane proteins, and we report here that the localization of Yck2p within the membrane is dynamic through the cell cycle. Using a functional green fluorescent protein (GFP) fusion, we have observed that Yck2p is concentrated at sites of polarized growth during bud morphogenesis. At cytokinesis, GFP–Yck2p becomes associated with a ring at the bud neck and then appears as a patch of fluorescence, apparently coincident with the dividing membranes. The bud neck association of Yck2p at cytokinesis does not require an intact septin ring, and septin assembly is altered in a Yck-deficient mutant. The sites of GFP–Yck2p concentration and the defects observed for Yck-deficient cells together suggest that Yck plays distinct roles in morphogenesis and cytokinesis that are effected by differential localization.

Phosphorylation of protein kinase N by phosphoinositide-dependent protein kinase-1 mediates insulin signals to the actin cytoskeleton

Growth factors such as insulin regulate phosphatidylinositol 3-kinase-dependent actin cytoskeleton rearrangement in many types of cells. However, the mechanism by which the insulin signal is transmitted to the actin cytoskeleton remains largely unknown. Yeast two-hybrid screening revealed that the phosphatidylinositol 3-kinase downstream effector phosphoinositide-dependent protein kinase-1 (PDK1) interacted with protein kinase N (PKN), a Rho-binding Ser/Thr protein kinase potentially implicated in a variety of cellular events, including phosphorylation of cytoskeletal components. PDK1 and PKN interacted in vitro and in intact cells, and this interaction was mediated by the kinase domain of PDK1 and the carboxyl terminus of PKN. In addition to a direct interaction, PDK1 also phosphorylated Thr774 in the activation loop and activated PKN. Insulin treatment or ectopic expression of the wild-type PDK1 or PKN, but not protein kinase Cζ, induced actin cytoskeleton reorganization and membrane ruffling in 3T3-L1 fibroblasts and Rat1 cells that stably express the insulin receptor (Rat1-IR). However, the insulin-stimulated actin cytoskeleton reorganization in Rat1-IR cells was prevented by expression of kinase-defective PDK1 or PDK1-phosphorylation site-mutated PKN. Thus, phosphorylation by PDK1 appears to be necessary for PKN to transduce signals from the insulin receptor to the actin cytoskeleton.

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.

Drosophila phosphoinositide-dependent kinase-1 regulates apoptosis and growth via the phosphoinositide 3-kinase-dependent signaling pathway

Phosphoinositide-dependent kinase-1 (PDK-1) is a central mediator of the cell signaling between phosphoinositide 3-kinase (PI3K) and various intracellular serine/threonine kinases including Akt/protein kinase B (PKB), p70 S6 kinases, and protein kinase C. Recent studies with cell transfection experiments have implied that PDK-1 may be involved in various cell functions including cell growth and apoptosis. However, despite its pivotal role in cellular signalings, the in vivo functions of PDK-1 in a multicellular system have rarely been investigated. Here, we have isolated Drosophila PDK-1 (dPDK-1) mutants and characterized the in vivo roles of the kinase. Drosophila deficient in the dPDK-1 gene exhibited lethality and an apoptotic phenotype in the embryonic stage. Conversely, overexpression of dPDK-1 increased cell and organ size in a Drosophila PI3K-dependent manner. dPDK-1 not only could activate Drosophila Akt/PKB (Dakt1), but also substitute the in vivo functions of its mammalian ortholog to activate Akt/PKB. This functional interaction between dPDK-1 and Dakt1 was further confirmed through genetic analyses in Drosophila. On the other hand, cAMP-dependent protein kinase, which has been proposed as a possible target of dPDK-1, did not interact with dPDK-1. In conclusion, our findings provide direct evidence that dPDK-1 regulates cell growth and apoptosis during Drosophila development via the PI3K-dependent signaling pathway and demonstrate our Drosophila system to be a powerful tool for elucidating the in vivo functions and targets of PDK-1.

Raf-1 promotes cell survival by antagonizing apoptosis signal-regulating kinase 1 through a MEK–ERK independent mechanism

The Ser/Thr kinase Raf-1 is a protooncogene product that is a central component in many signaling pathways involved in normal cell growth and oncogenic transformation. Upon activation, Raf-1 phosphorylates mitogen-activated protein kinase kinase (MEK), which in turn activates mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERKs), leading to the propagation of signals. Depending on specific stimuli and cellular environment, the Raf-1–MEK–ERK cascade regulates diverse cellular processes such as proliferation, differentiation, and apoptosis. Here, we describe a MEK–ERK-independent prosurvival function of Raf-1. We found that Raf-1 interacts with the proapoptotic, stress-activated protein kinase ASK1 (apoptosis signal-regulating kinase 1) in vitro and in vivo. Deletion analysis localized the Raf-1 binding site to the N-terminal regulatory fragment of ASK1. This interaction allows Raf-1 to act independently of the MEK–ERK pathway to inhibit apoptosis. Furthermore, catalytically inactive forms of Raf-1 can mimic the wild-type effect, raising the possibility of a kinase-independent function of Raf-1. Thus, Raf-1 may promote cell survival through its protein–protein interactions in addition to its established MEK kinase function.

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.

Effect of Water Stress on Cell Division and Cdc2-Like Cell Cycle Kinase Activity in Wheat Leaves1

In wheat (Triticum aestivum) seedlings subjected to a mild water stress (water potential of −0.3 MPa), the leaf-elongation rate was reduced by one-half and the mitotic activity of mesophyll cells was reduced to 42% of well-watered controls within 1 d. There was also a reduction in the length of the zone of mesophyll cell division to within 4 mm from the base compared with 8 mm in control leaves. However, the period of division continued longer in the stressed than in the control leaves, and the final cell number in the stressed leaves reached 85% of controls. Cyclin-dependent protein kinase enzymes that are required in vivo for DNA replication and mitosis were recovered from the meristematic zone of leaves by affinity for p13suc1. Water stress caused a reduction in H1 histone kinase activity to one-half of the control level, although amounts of the enzyme were unaffected. Reduced activity was correlated with an increased proportion of the 34-kD Cdc2-like kinase (an enzyme sharing with the Cdc2 protein of other eukaryotes the same size, antigenic sites, affinity for p13suc1, and H1 histone kinase catalytic activity) deactivated by tyrosine phosphorylation. Deactivation to 50% occurred within 3 h of stress imposition in cells at the base of the meristematic zone and was therefore too fast to be explained by a reduction in the rate at which cells reached mitosis because of slowing of growth; rather, stress must have acted more immediately on the enzyme. The operation of controls slowing the exit from the G1 and G2 phases is discussed. We suggest that a water-stress signal acts on Cdc2 kinase by increasing phosphorylation of tyrosine, causing a shift to the inhibited form and slowing cell production.

Activation of NF-κB by nontypeable Hemophilus influenzae is mediated by toll-like receptor 2-TAK1-dependent NIK–IKKα/β–IκBα and MKK3/6–p38 MAP kinase signaling pathways in epithelial cells

Nontypeable Hemophilus influenzae (NTHi) is an important human pathogen in both children and adults. In children, it causes otitis media, the most common childhood infection and the leading cause of conductive hearing loss in the United States. In adults, it causes lower respiratory tract infections in the setting of chronic obstructive pulmonary disease, the fourth leading cause of death in the United States. The molecular mechanisms underlying the pathogenesis of NTHi-induced infections remain undefined, but they may involve activation of NF-κB, a transcriptional activator of multiple host defense genes involved in immune and inflammatory responses. Here, we show that NTHi strongly activates NF-κB in human epithelial cells via two distinct signaling pathways, NF-κB translocation-dependent and -independent pathways. The NF-κB translocation-dependent pathway involves activation of NF-κB inducing kinase (NIK)–IKKα/β complex leading to IκBα phosphorylation and degradation, whereas the NF-κB translocation-independent pathway involves activation of MKK3/6–p38 mitogen-activated protein (MAP) kinase pathway. Bifurcation of NTHi-induced NIK–IKKα/β-IκBα and MKK3/6–p38 MAP kinase pathways may occur at transforming growth factor-β activated kinase 1 (TAK1). Furthermore, we show that toll-like receptor 2 (TLR2) is required for NTHi-induced NF-κB activation. In addition, several key inflammatory mediators including IL-1β, IL-8, and tumor necrosis factor-α are up-regulated by NTHi. Finally, P6, a 16-kDa lipoprotein highly conserved in the outer membrane of all NTHi and H. influenzae type b strains, appears to also activate NF-κB via similar signaling pathways. Taken together, our results demonstrate that NTHi activates NF-κB via TLR2–TAK1-dependent NIK–IKKα/β-IκBα and MKK3/6–p38 MAP kinase signaling pathways. These studies may bring new insights into molecular pathogenesis of NTHi-induced infections and open up new therapeutic targets for these diseases.