Rac-1 and IQGAP are potential regulators of E-cadherin-catenin interactions during murine preimplantation development

Adherens junction formation is fundamental for compaction and trophectoderm differentiation during mammalian preimplantation development. We recently isolated an IQGAP-2 cDNA from a differential display-polymerase chain reaction screen of bovine preimplantation developmental stages. IQGAP-1 and -2 proteins mediate E-cadherin-based cell-to-cell adhesion through interactions with beta-catenin and the Rho GTPases, rac1 and cdc42. Our study demonstrates IQGAP-1,-2, rac-1 and cdc42 mRNAs are present throughout murine preimplantation development. IQGAP-1 and rac-1 protein distribution changes from predominantly plasma membrane associated to predominantly cytoplasmic as the embryo progresses through cleavage divisions and compaction to the blastocyst stage.     

Expression of ZO-1 and occludin at mRNA and protein level during preimplantation development of the pig parthenogenetic diploids

Expression of mRNAs and proteins of ZO-1 and occludin was analyzed in pig oocytes and parthenogenetic diploid embryos during preimplantation development using real-time RT-PCR, western blotting and immunocytochemistry. All germinal vesicle (GV) and metaphase (M)II oocytes and preimplantation embryos expressed mRNAs and proteins of ZO-1 and occludin. mRNA levels of both ZO-1 and occludin decreased significantly from GV to MII, but increased at the 2-cell stage followed by temporal decrease during the early and late 4-cell stages. Then, both mRNAs increased after compaction. Relative concentration of zo1α- was highest in 2-cell embryos, while zo1α+ was expressed from the morula stage. Occludin expression greatly increased after the morula stage and was highest in expanded blastocysts. Western blotting analysis showed constant expression of ZO-1α- throughout preimplantation development and limited translation of ZO-1α+ from the blastocysts, and species-specific expression pattern of occludin. Immunocytochemistry analysis revealed homogeneous distribution of ZO-1 and occludin in the cytoplasm with moderately strong fluorescence in the vicinity of the contact region between blastomeres, around the nuclei in the 2-cell to late 4-cell embryos, and clear network localization along the cell-boundary region in embryos after the morula stage. Present results show that major TJ proteins, ZO-1 and occludin are expressed in oocytes and preimplantation embryos, and that ZO-1α+ is transcribed by zygotic gene activation and translated from early blastocysts with prominent increase of occludin at the blastocyst stage.     

Mutational analysis of CDC42Sc, a Saccharomyces cerevisiae gene that encodes a putative GTP-binding protein involved in the control of cell polarity.

The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity. We have analyzed the effects of three CDC42 mutations (Gly to Val-12, Gln to Leu-61, and Asp to Ala-118) in the putative GTP-binding and hydrolysis domains and one mutation (Cys to Ser-188) in the putative isoprenylation site. The first three mutations resulted in either a dominant-lethal or dose-dependent dominant-lethal phenotype when present on plasmids in haploid cdc42-1ts or wild-type strains. Both wild-type and cdc42-1ts cells carrying plasmids (pGAL) with either the CDC42Val-12 or CDC42Leu-61 alleles under the control of a GAL promoter were arrested with a novel phenotype of large cells with elongated or multiple buds. Cells carrying pGAL-CDC42Ala-118 were arrested as large, round, unbudded cells reminiscent of cdc42-1ts arrested cells. The different phenotype of the CDC42Ala-118 mutant versus the CDC42Val-12 and CDC42Leu-61 mutants was unexpected since the phenotypes of all three analogous ras mutants were similar to each other. This suggests that aspects of the biochemical properties of the Cdc42 protein differ from those of the Ras protein. The cdc42Ser-188 mutant gene was incapable of complementing the cdc42-1ts mutation and was recessive to both wild-type and cdc42-1ts. In double-mutant alleles, the cdc42Ser-188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.     

Role of cellular phosphatase cdc25C in herpes simplex virus 1 replication

Earlier studies have shown that in herpes simplex virus 1-infected cells, ICP22 upregulates the accumulation of a subset of gamma(2) proteins exemplified by the products of the U(L)38, U(L)41, and U(S)11 genes. The ICP22-dependent process involves degradation of cyclins A and B1, the stabilization and activation of cdc2, physical interaction of activated cdc2 with the U(L)42 DNA synthesis processivity factor, and recruitment and phosphorylation of topoisomerase IIalpha by the cdc2/U(L)42 complex. Activation of cdc2, the first step in the process, is a key function of the mitotic phosphatase cdc25C. To define the role of cdc25C, we probed some features of the ICP22-dependent pathway of upregulation of gamma(2) genes in cdc25C(-/-) cells and in cdc25C(+/+) cells derived from sibling mice. We report that cyclin B1 turned over in cdc25C(+/+) or cdc25C(-/-) cells at the same rate, that cdc2 increased in amount, and that U(S)11 and U(L)38 proteins and infectious virus accumulated in smaller amounts than in wild-type infected cells. The reduction in U(L)38 protein accumulation and virus was greater in cdc25C(-/-) cells infected with virus lacking ICP22 than in cells infected with wild-type virus. We conclude that cdc25C phosphatase plays a role in viral replication and that this role extends beyond its function of activating cdc2 for initiation of the ICP22-dependent cascade for upregulation of gamma(2) gene expression.     

Msb1 Interacts with Cdc42, Boi1, and Boi2 and May Coordinate Cdc42 and Rho1 Functions during Early Stage of Bud Development in Budding Yeast

Msb1 is not essential for growth in the budding yeast Saccharomyces cerevisiae since msb1Δ cells do not display obvious phenotypes. Genetic studies suggest that Msb1 positively regulates Cdc42 function during bud development, since high-copy MSB1 suppressed the growth defect of temperature-sensitive cdc24 and cdc42 mutants at restrictive temperature, while deletion of MSB1 showed synthetic lethality with cdc24, bem1, and bem2 mutations. However, the mechanism of how Msb1 regulates Cdc42 function remains poorly understood. Here, we show that Msb1 localizes to sites of polarized growth during bud development and interacts with Cdc42 in the cells. In addition, Msb1 interacts with Boi1 and Boi2, two scaffold proteins that also interact with Cdc42 and Bem1. These findings suggest that Msb1 may positively regulate Cdc42 function by interacting with Cdc42, Boi1, and Boi2, which may promote the efficient assembly of Cdc42, Cdc24, and other proteins into a functional complex. We also show that Msb1 interacts with Rho1 in the cells and Msb1 overproduction inhibits the growth of rho1-104 and rho1-3 but not rho1-2 cells. The growth inhibition appears to result from the down-regulation of Rho1 function in glucan synthesis, specifically during early stage of bud development. These results suggest that Msb1 may coordinate Cdc42 and Rho1 functions during early stage of bud development by promoting Cdc42 function and inhibiting Rho1 function. Msb1 overproduction also affects cell morphology, septin organization, and causes increased, aberrant deposition of 1,3-β-glucan and chitin at the mother-bud neck. However, the stimulation of glucan synthesis mainly occurs during late, but not early, stage of bud development.     

Inhibition of cytochrome P450 mediated enzyme activity by alkylphosphocholines

The inhibitory potency of four alkylphospholipids: rac-1-O-phosphocholine-2-hydroxy-octadecane (rac-2-OH), rac-1-O-phosphocholine-2-O-acetyl-octadecane (rac-2-O-acetyl), rac-1-O-phosphocholine-2-amino-octadecane (rac-2-NH2) and rac-1-O-phosphocholine-2-N-acetyloctadecane (rac-2-N-acetyl), on the cytochrome P450-dependent monooxygenase activity has been evaluated. The IC50 values of the alkylphosphocholines with 7-ethoxycoumarin as substrate in liver microsomal fractions of PB-treated rats and with a reconstituted CYP2B1: NADPH-P450-reductase system are in the range of 3.2-5.0 microM and 2.8-3.5 microM, respectively. Lineweaver-Burk plots with the inhibitors in concentrations that were found to cause roughly a 50% inhibition and with 7-ethoxycoumarin as substrate revealed for all four alkylphospholipids a competitive inhibition type. The degree of the competitive inhibition is quantified by the Ki values. With liver microsomal fractions of PB-treated rats, the Ki values of rac-2-OH (Ki = 1.36 microM) and rac-2-O-acetyl (Ki = 1.33 microM) differs slightly from those of rac-2-NH2 (Ki = 2.2 microM) and rac-2-N-acetyl (Ki = 2.2 microM), but with the reconstituted CYP2B1: NADPH-P450-reductase system all Ki values are in the small range of 1.8 - 2.6 microM, indicating that the short substituted group at the 2-position (OH; O-acetyl; NH2; N-acetyl) of the long chain octadecanol part of the phosphodiesters exhibit no essential role on the strong inhibitory potency of these alkylphosphocholines on the 7-ethoxycoumarin-O-deethylase activity.     

Cdc42 stimulates neurite outgrowth and formation of growth cone filopodia and lamellipodia

To assess the role of cdc42 during neurite development, cmyc-tagged constitutively active (CA) and dominant negative (DN) cdc42 were expressed in dissociated primary chick spinal cord neurons using adenoviral-mediated gene transfer. Three days after infection, >85% of the neurons in infected cultures expressed cdc42 proteins, as detected by indirect immunofluorescence against cmyc. Growth cones of infected neurons displayed 1.83- (CAcdc42) and 1.93-fold (DNcdc42) higher cmyc immunofluorescence per square micrometer than uninfected controls. CAcdc42 expression stimulated growth cones, almost doubling growth cone size and number of filopodia, and increased neurite growth rates by 65-89%. In neurons plated onto fibronectin, the percent of growth cones with both filopodia and lamellipodia increased from 71 to 92%. Total Texas Red-phalloidin staining in these growth cones doubled, and the percent of growth cones with F-actin localized to peripheral regions increased from 52% in controls to 78% after CAcdc42 expression. Expression of DNcdc42 did not significantly alter growth cone morphology or neurite growth rates. Addition of soluble laminin to spinal cord neurons resulted in the identical phenotype as CAcdc42 expression, including changes in growth cone morphology, F-actin localization, and neurite growth rates. Significantly, expression of DNcdc42 blocked the effects of laminin on growth cones. These results show that cdc42 promotes neurite outgrowth and filopodial and lamellipodial formation in growth cones and suggests that cdc42 and laminin share a common signaling pathway during neurite development. Addition of laminin to CAcdc42-expressing neurons is inhibitory to growth cones, indicating that laminin also may activate some other pathways.     

Presence of cdc2(+)-like proteins in the preimplantation mouse embryo

An antibody raised against a portion of the human equivalent of the yeast cdc2+ protein reacts with a 34K protein in mouse cell lines and early embryonic cells. Western blot analysis coupled with phosphatase treatment of material collected from the early preimplantation embryo has shown that the murine cdc2+ homologue does not correspond to the previously described newly synthesised proteins that are phosphorylated in a cell-cycle-dependent fashion [Howlett, 1986]. The cdc2(+)-like protein is converted into a slower migrating form on entry into S-phase and is further modified during G2 prior to mitosis. Studies of embryos that are held in extended periods of M-phase, i.e. unfertilised eggs or 1-cell embryos treated with nocodazole, demonstrate that the cdc2(+)-like protein becomes demodified in these cells.     

Compaction in preimplantation mouse embryos is regulated by a cytoplasmic regulatory factor that alters between 1- and 2-cell stages in a concentration-dependent manner.

Present studies were performed to investigate what factors affect the morphogenesis of preimplantation mouse embryos, and to find the action mechanism of that factor by using cytoplasm removal and its reconstitution from a different developmental stage embryo. Half (HP group) or one-third of cytoplasm (TP group) was removed from 1-cell mouse embryos by micromanipulation, and their morphogenesis and genome expression were compared with sham-operated embryos (SP group). The compaction and blastocoel formation of embryos in both the HP and TP groups were accelerated in time and cell stage when compared with those of the SP group. However, the total activity and time of RNA synthesis, and gene expression of ZO-1alpha+ isoform were not different. To change the cytoplasm composition without altering the nucleus/cytoplasmic ratio, half a 1-cell embryo with both pronuclei was reconstituted with the half enucleated cytoplasm of 1-cell embryo (P + P group), 2-cell (P + 2 group) or 4-cell (P + 4 group) by electrofusion. Embryonic compaction, timing of RNA synthesis, and stage-specific gene expression of the ZO-1alpha(+) isoform in the P + 2 and P + 4 groups were accelerated in time and cell stage than that in the P + P group, but not different between the P + 2 and P + 4 groups. In addition, a blastomere of 2-cell embryo was reconstituted with the enucleated cytoplasm of 1-cell embryo (2 + P group) or 2-cell (2 + 2 group) in equal volume by electrofusion. Also, the karyoplast of 2-cell was fused with the enucleated 1-cell embryo (2 + PP group). Embryonic development, total activity of RNA synthesis, and gene expression of the ZO-1alpha(+) isoform of embryos in the 2 + P and 2 + PP groups were delayed when compared with those of the 2 + 2 group. Also, the phenomena of compaction and blastocoel formation were delayed in the development time and cell stage. From these results, the nucleus/cytoplasm ratio was found to have no direct effect on the regulation of embryonic morphogenesis, although it accelerated compaction and blastocoel formation. However, cytoplasmic factors that altered between 1- and 2-cell stages regulate embryonic morphogenesis, especially compaction, of preimplantation mouse embryos in concentration-dependent manner.     

Stf1: Non-Wee Mutations Epistatic to Cdc25 in the Fission Yeast Schizosaccharomyces Pombe

In Schizosaccharomyces pombe, cdc25 is a cell cycle regulated inducer of mitosis. wee1 and phenotypically wee alleles of cdc2 are epistatic to cdc25. Mutant alleles of a new locus, stf1 (suppressor of twenty-five), identified in a reversion analysis of conditionally lethal cdr1-76 cdc25-22 and cdr2-96 cdc25-22 double mutant strains, also suppress both temperature-sensitive and gene disruption alleles of cdc25. These mutants, by themselves, are phenotypically indistinguishable from wild type strains; hence they represent the first known mutations that are epistatic to cdc25 and do not display a wee phenotype. stf1 genetically interacts with other elements of mitotic control in S. pombe. stf1-1 is additive with wee1-50, cdc2-1w and cdc2-3w for suppression of cdc25-22. Also, like wee1- and cdc2-w, stf1- suppression of cdc25 is reversed by overexpression of the putative type 1 protein phosphatase bws1+/dis2+. Interaction with various mutants and plasmid overexpression experiments suggest that stf1 does not operate either upstream or downstream of wee1. Similarly, it does not operate through cdc25 since it rescues the disruption. stf1 appears to encode an important new element of mitotic control.     

Cdc4, a Protein Required for the Onset of S Phase, Serves an Essential Function during G2/M Transition in Saccharomyces cerevisiae

Saccharomyces cerevisiae proteins Cdc4 and Cdc20 contain WD40 repeats and participate in proteolytic processes. However, they are thought to act at two different stages of the cell cycle: Cdc4 is involved in the proteolysis of the Cdk inhibitor, Sic1, necessary for G(1)/S transition, while Cdc20 mediates anaphase-promoting complex-dependent degradation of anaphase inhibitor Pds1, a process necessary for the onset of chromosome segregation. We have isolated three mutant alleles of CDC4 (cdc4-10, cdc4-11, and cdc4-16) which suppress the nuclear division defect of cdc20-1 cells. However, the previously characterized mutation cdc4-1 and a new allele, cdc4-12, do not alleviate the defect of cdc20-1 cells. This genetic interaction suggests an additional role for Cdc4 in G(2)/M. Reexamination of the cdc4-1 mutant revealed that, in addition to being defective in the onset of S phase, it is also defective in G(2)/M transition when released from hydroxyurea-induced S-phase arrest. A second function for CDC4 in late S or G(2) phase was further confirmed by the observation that cells lacking the CDC4 gene are arrested both at G(1)/S and at G(2)/M. We subsequently isolated additional temperature-sensitive mutations in the CDC4 gene (such as cdc4-12) that render the mutant defective in both G(1)/S and G(2)/M transitions at the restrictive temperature. While the G(1)/S block in both cdc4-12 and cdc4Delta mutants is abolished by the deletion of the SIC1 gene (causing the mutants to be arrested predominantly in G(2)/M), the preanaphase arrest in the cdc4-12 mutant is relieved by the deletion of PDS1. Collectively, these observations suggest that, in addition to its involvement in the initiation of S phase, Cdc4 may also be required for the onset of anaphase.     

Rapid actions of plasma membrane estrogen receptors regulate motility of mouse embryonic stem cells through a profilin-1/cofilin-1-directed kinase signaling pathway

Long-term estrogen actions are vital for driving cell growth, but more recent evidence suggests that estrogen mediates more rapid cellular effects. However, the function of estradiol-17β (E(2))-BSA in mouse embryonic stem cells has not been reported. Therefore, we examined the role of E(2)-BSA in mouse embryonic stem cell motility and its related signal pathways. E(2)-BSA (10(-8) m) significantly increased motility after 24 h incubation and increased filamentous (F)-actin expression; these effects were inhibited by the estrogen receptor antagonist ICI 182,780, indicating that E(2)-BSA bound membrane estrogen receptors and initiated a signal. E(2)-BSA increased c-Src and focal adhesion kinase (FAK) phosphorylation, which was attenuated by ICI 182,780. The E(2)-BSA-induced increase in epidermal growth factor receptor (EGFR) phosphorylation was inhibited by Src inhibitor PP2. As a downstream signal molecule, E(2)-BSA activated cdc42 and increased formation of a complex with the neural Wiskott-Aldrich syndrome protein (N-WASP)/cdc42/transducer of cdc42-dependent actin assembly-1 (TOCA-1), which was inhibited by FAK small interfering RNA (siRNA) and EGFR inhibitor AG 1478. In addition, E(2)-BSA increased profilin-1 expression and cofilin-1 phosphorylation, which was blocked by cdc42 siRNA. Subsequently, E(2)-BSA induced an increase in F-actin expression, and cell motility was inhibited by each signal pathway-related siRNA molecule or inhibitors but not by cofilin-1 siRNA. A combined treatment of cofilin-1 siRNA and E(2)-BSA increased F-actin expression and cell motility more than that of E(2)-BSA alone. These data demonstrate that E(2)-BSA stimulated motility by interacting with profilin-1/cofilin-1 and F-actin through FAK- and c-Src/EGFR transactivation-dependent N-WASP/cdc42/TOCA-1 complex.     

Cdc42p GTPase is involved in controlling polarized cell growth in Schizosaccharomyces pombe.

Cdc42p is a highly conserved low-molecular-weight GTPase that is involved in controlling cellular morphogenesis. We have isolated the Cdc42p homolog from the fission yeast Schizosaccharomyces pombe by its ability to complement the Saccharomyces cerevisiae cdc42-1ts mutation. S. pombe Cdc42p is 85% identical in predicted amino acid sequence to S. cerevisiae Cdc42p and 83% identical to the human Cdc42p homolog. The Cdc42p protein fractionates to both soluble and particulate fractions, suggesting that it exists in two cellular pools. We have disrupted the cdc42+ gene and shown that it is essential for growth. The cdc42 null phenotype is an arrest as small, round, dense cells. In addition, we have generated three site-specific mutations, G12V, Q61L, and D118A, in the Cdc42p GTP-binding domains that correspond to dominant-lethal mutations in S. cerevisiae CDC42. In contrast to the S. cerevisiae cdc42 mutations, the S. pombe cdc42 mutant alleles were not lethal when overexpressed. However, the cdc42 mutants did exhibit an abnormal morphological phenotype of large, misshapen cells, suggesting that S. pombe Cdc42p is involved in controlling polarized cell growth.     

Activation of protein kinase C triggers premature compaction in the four-cell stage mouse embryo

During mouse preimplantation development, the cells of the mouse embryo undergo a progressive subcellular reorganization at compaction, which eventually results in the formation of two distinct cell types. We have investigated the effect that activators of the Ca2(+)-phospholipid-dependent protein kinase (PKC) have on mouse compaction. Phorbol ester activation of PKC caused premature compaction of four-cell embryos within a few minutes of addition followed by a prolonged decompaction phase after 1 hr. This response was dose-dependent to concentrations as low as 250 pg/ml. Diacylglycerides also caused compaction; however, it was more sustained than with phorbol esters and was not followed by a phase of decompaction. Inhibition of PKC with sphingosine blocks induced compaction in a dose-dependent manner and also blocks normal compaction of eight-cell embryos. A monoclonal antibody to the cell adhesion molecule, E-cadherin, which mediates mouse embryo compaction, completely blocks compaction induced by these activators of PKC. Indirect immunofluorescence with a monoclonal antibody to E-cadherin indicates that PKC activation causes a rapid shift in the localization of this cell adhesion molecule, which coincides with the observed compaction. These results suggest that PKC plays a role in the initiation of compaction through its effect either directly or indirectly on E-cadherin.     

Rho 1 GTPase activates the (1-3)beta-D-glucan synthase and is involved in Schizosaccharomyces pombe morphogenesis.

The Schizosaccharomyces pombe Cdc42 and Rho1 GTPases were tested for their ability to complement the cwg2-1 mutant phenotype of a decrease in (1-3)beta-D-glucan synthase activity when grown at the non-permissive temperature. Only Rho1 is able to partly complement the defect in glucan synthase associated with the cwg2-1 mutation. Moreover, overexpression of the rho1 gene in wild-type S.pombe cells causes aberrant morphology with loss of polarity and cells with several septa. Under this condition (1-3)beta-D-glucan synthase activity is increased four times, but is still dependent on GTP. When S.pombe is transformed with constitutively active rho1 mutant alleles (rho1-G15V or rho1-Q64L), cells stop growing and show a very thick cell wall with hardly any septum. Under this condition the level of (1-3)beta-D-glucan synthase activity is at least 20 times higher than wild-type and is independent of GTP. Neither cdc42+ nor the cdc42-V12G or cdc42-Q61L constitutively active mutant alleles affect (1-3)beta-D-glucan synthase activity when overexpressed in S.pombe. Cells overproducing Rho1 are hypersensitive to inhibitors of cell wall biosynthesis or to cell wall degrading enzymes. We conclude that Rho1 GTPase directly activates (1-3)beta-D-glucan synthase and regulates S.pombe morphogenesis.     

Cdc42 is a Rho GTPase family member that can mediate insulin signaling to glucose transport in 3T3-L1 adipocytes

We investigated the role of cdc42, a Rho GTPase family member, in insulin-induced glucose transport in 3T3-L1 adipocytes. Microinjection of anti-cdc42 antibody or cdc42 siRNA led to decreased insulin-induced and constitutively active G(q) (CA-G(q); Q209L)-induced GLUT4 translocation. Adenovirus-mediated expression of constitutively active cdc42 (CA-cdc42; V12) stimulated 2-deoxyglucose uptake to 56% of the maximal insulin response, and this was blocked by treatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin, or LY294002. Both insulin and CA-G(q) expression caused an increase in cdc42 activity, showing that cdc42 is activated by insulin and is downstream of G alpha(q/11) in this activation pathway. Immunoprecipitation experiments showed that insulin enhanced a direct association of cdc42 and p85, and both insulin treatment and CA-cdc42 expression stimulated PI3-kinase activity in immunoprecipitates with anti-cdc42 antibody. Furthermore, the effects of insulin, CA-G(q), and CA-cdc42 on GLUT4 translocation or 2-deoxyglucose uptake were inhibited by microinjection of anti-protein kinase C lambda (PKC lambda) antibody or overexpression of a kinase-deficient PKC lambda construct. In summary, activated cdc42 can mediate 1) insulin-stimulated GLUT4 translocation and 2) glucose transport in a PI3-kinase-dependent manner. 3) Insulin treatment and constitutively active G(q) expression can enhance the cdc42 activity state as well as the association of cdc42 with activated PI3-kinase. 4) PKC lambda inhibition blocks CA-cdc42, CA-G(q), and insulin-stimulated GLUT4 translocation. Taken together, these data indicate that cdc42 can mediate insulin signaling to GLUT4 translocation and lies downstream of G alpha(q/11) and upstream of PI3-kinase and PKC lambda in this stimulatory pathway.     

Selective control of basolateral membrane protein polarity by cdc42

The rho GTPase cdc42 is implicated in several aspects of cell polarity. A recent study (Kroschewski R, Hall A, Mellman I. Nat Cell Biol 1999;1:8–13) demonstrated that a dominant negative mutant of cdc42 abolishes the polarity of basolateral membrane proteins in MDCK cells, but did not elucidate whether this effect was selective for basolateral proteins or nonselective for all secreted proteins. To answer this question, we analyzed the polarity of newly synthesized membrane and soluble proteins in MDCK cell lines previously induced to overexpress mutant forms of cdc42. GTPase-deficient and dominant negative cdc42 did not affect the apical targeting of a newly synthesized apical membrane protein, but reversed to apical the distribution of two exogenous basolateral membrane proteins. In striking contrast, GTPase-deficient cdc42 did not affect polarized exocytosis of endogenous soluble proteins, either apical or basolateral. The exquisitely selective regulation of polarized protein targeting by cdc42 may allow cells to fine-tune their membrane composition in response to extracellular signals during development, migration and in response to injury.     

cdc2 cyclin-dependent kinase binds and phosphorylates herpes simplex virus 1 U(L)42 DNA synthesis processivity factor

Earlier studies have shown that cdc2 kinase is activated during herpes simplex virus 1 infection and that its activity is enhanced late in infection even though the levels of cyclin A and B are decreased below levels of detection. Furthermore, activation of cdc2 requires the presence of infected cell protein no. 22 and the U_L13 protein kinase, the same gene products required for optimal expression of a subset of late genes exemplified by U_S11, U_L38, and U_L41. The possibility that the activation of cdc2 and expression of this subset may be connected emerged from the observation that dominant negative cdc2 specifically blocked the expression of U_S11 protein in cells infected and expressing dominant negative cdc2. Here we report that in the course of searching for a putative cognate partner for cdc2 that may have replaced cyclins A and B, we noted that the DNA polymerase processivity factor encoded by the U_L42 gene contains a degenerate cyclin box and has been reported to be structurally related to proliferating cell nuclear antigen, which also binds cdk2. Consistent with this finding, we report that (i) U_L42 is able to physically interact with cdc2 at both the amino-terminal and carboxyl-terminal domains, (ii) the carboxyl-terminal domain of U_L42 can be phosphorylated by cdc2, (iii) immunoprecipitates obtained with anti U_L42 antibody contained a roscovitine-sensitive kinase activity, (iv) kinase activity associated with U_L42 could be immunodepleted by antibody to cdc2, and (v) U_L42 transfected into cells associates with a nocodazole-enhanced kinase. We conclude that U_L42 can associate with cdc2 and that the kinase activity has the characteristic traits of cdc2 kinase.