Inactivation and self -association of CaM kinase: Effects of ATP, substrate binding domains, and isozymic forms

Calcium/calmodulin-dependent protein kinase II (CaM kinase) is a multifunctional Ser/Thr protein kinase, that is highly enriched in brain and is involved in regulating many aspects of neuronal function. We observed that forebrain CaM kinase from crude homogenates, cytosolic fractions and purified preparations inactivates and translocates into the particulate fraction following autophosphorylation. Using purified forebrain CaM kinase as well as recombinant $\alpha$ isozyme, we determined that the formation of particulate enzyme was due to enzyme self-association. The conditions of autophosphorylation determine whether enzyme self-association and/or inactivation will occur. Self-association of CaM kinase is sensitive to pH, ATP concentration, and enzyme autophosphorylation. This process is prevented by saturating concentrations of ATP. However, in limiting ATP, pH is the dominant factor, and enzyme self-association occurs at pH values $\rm{<}7.0.$ Site-specific mutants were produced by substituting Ala for Thr286, Thr253, or Thr305,306 to determine whether these sites of autophosphorylation affect enzyme inactivation and self-association. The only mutation that influenced these processes was Ala286, which removed the protective effect afforded by autophosphorylation in saturating ATP. Enzyme inactivation occurs in the presence and absence of self-association and appears predominantly sensitive to nucleotide concentration, because saturating concentrations of $\rm Mg\sp{2+}/ADP$ or $\rm Mg\sp{2+}/ATP$ prevent this process. These data implicate the ATP binding pocket in both inactivation and self-association. We also observed that select peptide substrates and peptide inhibitors modeled after the autoregulatory domain of CaM kinase prevented these processes. The $\alpha$ and $\beta$ isozymes of CaM kinase were characterized independently, and were observed to exhibit differences in both enzyme inactivation and self-association. The $\beta$ isozyme was less sensitive to inactivation, and was never observed to self-associate. Biophysical characterization, and transmission electron microscopy coupled with image analysis indicated both isozymes were multimeric, however, the $\alpha$ and $\beta$ isozymes appeared structurally different. We hypothesize that the $\alpha$ subunit of CaM kinase plays both a structural and enzymatic role, and the $\beta$ subunit plays an enzymatic role. The ramifications for the functional differences observed for inactivation and self-association are discussed based on potential structural differences and autoregulation of the $\alpha$ and $\beta$ isozymes in both calcium-induced physiological and pathological processes. ^

Inactivation of the mitogen-activated protein kinase Mps1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defense responses

The rice blast fungus, Magnaporthe grisea, generates enormous turgor pressure within a specialized cell called the appressorium to breach the surface of host plant cells. Here, we show that a mitogen-activated protein kinase, Mps1, is essential for appressorium penetration. Mps1 is 85% similar to yeast Slt2 mitogen-activated protein kinase and can rescue the thermosensitive growth of slt2 null mutants. The mps1–1Δ mutants of M. grisea have some phenotypes in common with slt2 mutants of yeast, including sensitivity to cell-wall-digesting enzymes, but display additional phenotypes, including reduced sporulation and fertility. Interestingly, mps1–1Δ mutants are completely nonpathogenic because of the inability of appressoria to penetrate plant cell surfaces, suggesting that penetration requires remodeling of the appressorium wall through an Mps1-dependent signaling pathway. Although mps1–1Δ mutants are unable to cause disease, they are able to trigger early plant-cell defense responses, including the accumulation of autofluorescent compounds and the rearrangement of the actin cytoskeleton. We conclude that MPS1 is essential for pathogen penetration; however, penetration is not required for induction of some plant defense responses.

In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis

We have developed a technique, methylation-specific PCR in situ hybridization (MSP-ISH), which allows for the methylation status of specific DNA sequences to be visualized in individual cells. We use MSP-ISH to monitor the timing and consequences of aberrant hypermethylation of the p16 tumor suppresser gene during the progression of cancers of the lung and cervix. Hypermethylation of p16 was localized only to the neoplastic cells in both in situ lesions and invasive cancers, and was associated with loss of p16 protein expression. MSP-ISH allowed us to dissect the surprising finding that p16 hypermethylation occurs in cervical carcinoma. This tumor is associated with infection of the oncogenic human papillomavirus, which expresses a protein, E7, that inactivates the retinoblastoma (Rb) protein. Thus, simultaneous Rb and p16 inactivation would not be needed to abrogate the critical cyclin D–Rb pathway. MSP-ISH reveals that p16 hypermethylation occurs heterogeneously within early cervical tumor cell populations that are separate from those expressing viral E7 transcripts. In advanced cervical cancers, the majority of cells have a hypermethylated p16, lack p16 protein, but no longer express E7. These data suggest that p16 inactivation is selected as the most effective mechanism of blocking the cyclin D–Rb pathway during the evolution of an invasive cancer from precursor lesions. These studies demonstrate that MSP-ISH is a powerful approach for studying the dynamics of aberrant methylation of critical tumor suppressor genes during tumor evolution.

A first-generation X-inactivation profile of the human X chromosome

In females, most genes on the X chromosome are generally assumed to be transcriptionally silenced on the inactive X as a result of X inactivation. However, particularly in humans, an increasing number of genes are known to “escape” X inactivation and are expressed from both the active (Xa) and inactive (Xi) X chromosomes; such genes reflect different molecular and epigenetic responses to X inactivation and are candidates for phenotypes associated with X aneuploidy. To identify genes that escape X inactivation and to generate a first-generation X-inactivation profile of the X, we have evaluated the expression of 224 X-linked genes and expressed sequence tags by reverse-transcription–PCR analysis of a panel of multiple independent mouse/human somatic cell hybrids containing a normal human Xi but no Xa. The resulting survey yields an initial X-inactivation profile that is estimated to represent ≈10% of all X-linked transcripts. Of the 224 transcripts tested here, 34 (three of which are pseudoautosomal) were expressed in as many as nine Xi hybrids and thus appear to escape inactivation. The genes that escape inactivation are distributed nonrandomly along the X; 31 of 34 such transcripts map to Xp, implying that the two arms of the X are epigenetically and/or evolutionarily distinct and suggesting that genetic imbalance of Xp may be more severe clinically than imbalance of Xq. A complete X-inactivation profile will provide information relevant to clinical genetics and genetic counseling and should yield insight into the genomic and epigenetic organization of the X chromosome.

HSP70 homolog functions in cell-to-cell movement of a plant virus

Plant closteroviruses encode a homolog of the HSP70 (heat shock protein, 70 kDa) family of cellular proteins. To facilitate studies of the function of HSP70 homolog (HSP70h) in viral infection, the beet yellows closterovirus (BYV) was modified to express green fluorescent protein. This tagged virus was competent in cell-to-cell movement, producing multicellular infection foci similar to those formed by the wild-type BYV. Inactivation of the HSP70h gene by replacement of the start codon or by deletion of 493 codons resulted in complete arrest of BYV translocation from cell to cell. Identical movement-deficient phenotypes were observed in BYV variants possessing HSP70h that lacked the computer-predicted ATPase domain or the C-terminal domain, or that harbored point mutations in the putative catalytic site of the ATPase. These results demonstrate that the virus-specific member of the HSP70 family of molecular chaperones functions in intercellular translocation and represents an additional type of a plant viral-movement protein.

Transforming growth factor β targeted inactivation of cyclin E:cyclin-dependent kinase 2 (Cdk2) complexes by inhibition of Cdk2 activating kinase activity

Transforming growth factor β (TGF-β)-mediated G1 arrest previously has been shown to specifically target inactivation of cyclin D:cyclin-dependent kinase (Cdk) 4/6 complexes. We report here that TGF-β-treated human HepG2 hepatocellular carcinoma cells arrest in G1, but retain continued cyclin D:Cdk4/6 activity and active, hypophosphorylated retinoblastoma tumor suppressor protein. Consistent with this observation, TGF-β-treated cells failed to induce p15INK4b, down-regulate CDC25A, or increase levels of p21CIP1, p27KIP1, and p57KIP2. However, TGF-β treatment resulted in the specific inactivation of cyclin E:Cdk2 complexes caused by absence of the activating Thr160 phosphorylation on Cdk2. Whole-cell lysates from TGF-β-treated cells showed inhibition of Cdk2 Thr160 Cdk activating kinase (CAK) activity; however, cyclin H:Cdk7 activity, a previously assumed mammalian CAK, was not altered. Saccharomyces cerevisiae contains a genetically and biochemically proven CAK gene, CAK1, that encodes a monomeric 44-kDa Cak1p protein unrelated to Cdk7. Anti-Cak1p antibodies cross-reacted with a 45-kDa human protein with CAK activity that was specifically down-regulated in response to TGF-β treatment. Taken together, these observations demonstrate that TGF-β signaling mediates a G1 arrest in HepG2 cells by targeting Cdk2 CAK and suggests the presence of at least two mammalian CAKs: one specific for Cdk2 and one for Cdk4/6.

Mössbauer spectroscopy as a tool for the study of activation/inactivation of the transcription regulator FNR in whole cells of Escherichia coli

The global regulator FNR (for fumarate nitrate reduction) controls the transcription of >100 genes whose products facilitate adaptation of Escherichia coli to growth under O2-limiting conditions. Previous Mössbauer studies have shown that anaerobically purified FNR contains a [4Fe-4S]2+ cluster that, on exposure to oxygen, is converted into a [2Fe-2S]2+ cluster, a process that decreases DNA binding by FNR. Using 57Fe Mössbauer spectroscopy of E. coli cells containing overexpressed FNR, we show here that the same cluster conversion also occurs in vivo on exposure to O2. Furthermore, the data show that a significant amount of the [4Fe-4S]2+ cluster is regenerated when the cells are shifted back to an anaerobic environment. The present study also demonstrates that 57Fe Mössbauer spectroscopy can be employed to study the in vivo behavior of (overexpressed) proteins. The use of this technique to study other iron-containing cell components is discussed.

Hair cell-specific splicing of mRNA for the α1D subunit of voltage-gated Ca2+ channels in the chicken’s cochlea

The L-type voltage-gated Ca2+ channels that control tonic release of neurotransmitter from hair cells exhibit unusual electrophysiological properties: a low activation threshold, rapid activation and deactivation, and a lack of Ca2+-dependent inactivation. We have inquired whether these characteristics result from cell-specific splicing of the mRNA for the L-type α1D subunit that predominates in hair cells of the chicken’s cochlea. The α1D subunit in hair cells contains three uncommon exons: one encoding a 26-aa insert in the cytoplasmic loop between repeats I and II, an alternative exon for transmembrane segment IIIS2, and a heretofore undescribed exon specifying a 10-aa insert in the cytoplasmic loop between segments IVS2 and IVS3. We propose that the alternative splicing of the α1D mRNA contributes to the unusual behavior of the hair cell’s voltage-gated Ca2+ channels.

Inactivation of calcium-activated chloride channels in smooth muscle by calcium/calmodulin-dependent protein kinase

To determine the mechanisms responsible for the termination of Ca2+-activated Cl− currents (ICl(Ca)), simultaneous measurements of whole cell currents and intracellular Ca2+ concentration ([Ca2+]i) were made in equine tracheal myocytes. In nondialyzed cells, or cells dialyzed with 1 mM ATP, ICl(Ca) decayed before the [Ca2+]i decline, whereas the calcium-activated potassium current decayed at the same rate as [Ca2+]i. Substitution of AMP-PNP or ADP for ATP markedly prolonged the decay of ICl(Ca), resulting in a rate of current decay similar to that of the fall in [Ca2+]i. In the presence of ATP, dialysis of the calmodulin antagonist W7, the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor KN93, or a CaMKII-specific peptide inhibitor the rate of ICl(Ca) decay was slowed and matched the [Ca2+]i decline, whereas H7, a nonspecific kinase inhibitor with low affinity for CaMKII, was without effect. When a sustained increase in [Ca2+]i was produced in ATP dialyzed cells, the current decayed completely, whereas in cells loaded with 5′-adenylylimidodiphosphate (AMP-PNP), KN93, or the CaMKII inhibitory peptide, ICl(Ca) did not decay. Slowly decaying currents were repeatedly evoked in ADP- or AMP-PNP-loaded cells, but dialysis of adenosine 5′-O-(3-thiotriphosphate) or okadaic acid resulted in a smaller initial ICl(Ca), and little or no current (despite a normal [Ca2+]i transient) with a second stimulation. These data indicate that CaMKII phosphorylation results in the inactivation of calcium-activated chloride channels, and that transition from the inactivated state to the closed state requires protein dephosphorylation.

Liz1p, a Novel Fission Yeast Membrane Protein, Is Required for Normal Cell Division When Ribonucleotide Reductase Is Inhibited

Ribonucleotide reductase activity is required for generating deoxyribonucleotides for DNA replication. Schizosaccharomyces pombe cells lacking ribonucleotide reductase activity arrest during S phase of the cell cycle. In a screen for hydroxyurea-sensitive mutants in S. pombe, we have identified a gene, liz1+, which when mutated reveals an additional, previously undescribed role for ribonucleotide reductase activity during mitosis. Inactivation of ribonucleotide reductase, by either hydroxyurea or a cdc22-M45 mutation, causes liz1− cells in G2 to undergo an aberrant mitosis, resulting in chromosome missegregation and late mitotic arrest. liz1+ encodes a 514-amino acid protein with strong similarity to a family of transmembrane transporters, and localizes to the plasma membrane of the cell. These results reveal an unexpected G2/M function of ribonucleotide reductase and establish that defects in a transmembrane protein can affect cell cycle progression.

Inhibition of Rho Is Required for cAMP-induced Melanoma Cell Differentiation

Up-regulation of the cAMP pathway by forskolin or α-melanocyte stimulating hormone induces melanocyte and melanoma cell differentiation characterized by stimulation of melanin synthesis and dendrite development. Here we show that forskolin-induced dendricity is associated to a disassembly of actin stress fibers. Since Rho controls actin organization, we studied the role of this guanosine triphosphate (GTP)-binding protein in cAMP-induced dendrite formation. Clostridium botulinum C3 exotransferase, which inhibits Rho, mimicked the effect of forskolin in promoting dendricity and stress fiber disruption, while the Escherichia coli toxin cytotoxic necrotizing factor-1 (CNF-1), which activates Rho and the expression of a constitutively active Rho mutant, blocked forskolin-induced dendrite outgrowth. In addition, overexpression of a constitutively active form of the Rho target p160 Rho-kinase (P160ROCK) prevented the dendritogenic effects of cAMP. Our results suggest that inhibition of Rho and of its target p160ROCK are required events for cAMP-induced dendrite outgrowth in B16 cells. Furthermore, we present evidence that Rho is involved in the regulation of melanogenesis. Indeed, Rho inactivation enhanced the cAMP stimulation of tyrosinase gene transcription and protein expression, while Rho constitutive activation impaired these cAMP-induced effects. This reveals that, in addition to controlling dendricity, Rho also participates in the regulation of melanin synthesis by cAMP.

Na–H Exchange Acts Downstream of RhoA to Regulate Integrin-induced Cell Adhesion and Spreading

The ubiquitously expressed Na–H exchanger NHE1 functions in regulating intracellular pH and cell volume. NHE1 activity is stimulated by hormones, growth factors, and activation of integrin receptors. We recently determined that NHE1 activity is also stimulated by activation of the low molecular weight GTPase RhoA and that increases in NHE1 activity are necessary for RhoA-induced formation of actin stress fibers. We now show that NHE1 acts downstream of RhoA to modulate initial steps in integrin signaling for the assembly of focal adhesions. Adhesion of CCL39 fibroblasts on fibronectin was markedly delayed in the presence of the NHE inhibitor ethylisopropylamiloride. In mutant PS120 cells, derived from CCL39 fibroblasts but lacking NHE1, adhesion was also delayed but was rescued in PS120 cells stably expressing NHE1. In the absence of NHE1 activity, cell spreading was inhibited, and the accumulation of integrins, paxillin, and vinculin at focal contacts was impaired. Additionally, tyrosine phosphorylation of p125FAK induced by integrin clustering was also impaired. Inactivation of RhoA with C3 transferase and inhibition of the Rho-kinase p160ROCK with the pyridine derivative Y-27632 completely abolished activation of NHE1 by integrins but not by platelet-derived growth factor. These findings indicate that NHE1 acts downstream of RhoA to contribute a previously unrecognized critical signal to proximal events in integrin-induced cytoskeletal reorganization.

Syntaxin Is Required for Cell Division

We recently identified a single family member homologue of syntaxin in the sea urchin. Syntaxin is present throughout development, and in rapidly dividing cleavage stage embryos it is present on numerous vesicles at the cell cortex. We hypothesized that syntaxin mediates essential membrane fusion events during early embryogenesis, reasoning that the vesicles and/or their contents are important for development. Here we show that functional inactivation of syntaxin with either Botulinum neurotoxin C1, which specifically proteolyzes syntaxin, or antibodies against syntaxin results in an inhibition of cell division. These observations suggest that syntaxin is essential for membrane fusion events critical for cell division.

A WD Repeat Protein Controls the Cell Cycle and Differentiation by Negatively Regulating Cdc2/B-Type Cyclin Complexes

In the fission yeast Schizosaccharomyces pombe, p34cdc2 plays a central role controlling the cell cycle. We recently isolated a new gene named srw1+, capable of encoding a WD repeat protein, as a multicopy suppressor of hyperactivated p34cdc2. Cells lacking srw1+ are sterile and defective in cell cycle controls. When starved for nitrogen source, they fail to effectively arrest in G1 and die of accelerated mitotic catastrophe if regulation of p34cdc2/Cdc13 by inhibitory tyrosine phosphorylation is compromised by partial inactivation of Wee1 kinase. Fertility is restored to the disruptant by deletion of Cig2 B-type cyclin or slight inactivation of p34cdc2. srw1+ shares functional similarity with rum1+, having abilities to induce endoreplication and restore fertility to rum1 disruptants. In the srw1 disruptant, Cdc13 fails to be degraded when cells are starved for nitrogen. We conclude that Srw1 controls differentiation and cell cycling at least by negatively regulating Cig2- and Cdc13-associated p34cdc2 and that one of its roles is to down-regulate the level of the mitotic cyclin particularly in nitrogen-poor environments.

PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway

To investigate the molecular basis of PTEN-mediated tumor suppression, we introduced a null mutation into the mouse Pten gene by homologous recombination in embryonic stem (ES) cells. Pten−/− ES cells exhibited an increased growth rate and proliferated even in the absence of serum. ES cells lacking PTEN function also displayed advanced entry into S phase. This accelerated G1/S transition was accompanied by down-regulation of p27KIP1, a major inhibitor for G1 cyclin-dependent kinases. Inactivation of PTEN in ES cells and in embryonic fibroblasts resulted in elevated levels of phosphatidylinositol 3,4,5,-trisphosphate, a product of phosphatidylinositol 3 kinase. Consequently, PTEN deficiency led to dosage-dependent increases in phosphorylation and activation of Akt/protein kinase B, a well-characterized target of the phosphatidylinositol 3 kinase signaling pathway. Akt activation increased Bad phosphorylation and promoted Pten−/− cell survival. Our studies suggest that PTEN regulates the phosphatidylinositol 3,4,5,-trisphosphate and Akt signaling pathway and consequently modulates two critical cellular processes: cell cycle progression and cell survival.