Cln3-associated kinase activity in Saccharomyces cerevisiae is regulated by the mating factor pathway.

The Saccharomyces cerevisiae cell cycle is arrested in G1 phase by the mating factor pathway. Genetic evidence has suggested that the G1 cyclins Cln1, Cln2, and Cln3 are targets of this pathway whose inhibition results in G1 arrest. Inhibition of Cln1- and Cln2-associated kinase activity by the mating factor pathway acting through Far1 has been described. Here we report that Cln3-associated kinase activity is inhibited by mating factor treatment, with dose response and timing consistent with involvement in cell cycle arrest. No regulation of Cln3-associated kinase was observed in a fus3 kss1 strain deficient in mating factor pathway mitogen-activated protein (MAP) kinases. Inhibition occurs mainly at the level of specific activity of Cln3-Cdc28 complexes. Inhibition of the C-terminally truncated Cln3-1-associated kinase is not observed; such truncations were previously identified genetically as causing resistance to mating factor-induced cell cycle arrest. Regulation of Cln3-associated kinase specific activity by mating factor treatment requires Far1. Overexpression of Far1 restores inhibition of C-terminally truncated Cln3-1-associated kinase activity. G2/M-arrested cells are unable to regulate Cln3-associated kinase, possibly because of cell cycle regulation of Far1 abundance. Inhibition of Cln3-associated kinase activity by the mating factor pathway may allow this pathway to block the earliest step in normal cell cycle initiation, since Cln3 functions as the most upstream G1-acting cyclin, activating transcription of the G1 cyclins CLN1 and CLN2 as well as of the S-phase cyclins CLB5 and CLB6.

Pheromone-dependent G1 cell cycle arrest requires Far1 phosphorylation, but may not involve inhibition of Cdc28-Cln2 kinase, in vivo.

In yeast, the pheromone alpha-factor acts as an antiproliferative factor that induces G1 arrest and cellular differentiation. Previous data have indicated that Far1, a factor dedicated to pheromone-induced cell cycle arrest, is under positive and negative posttranslational regulation. Phosphorylation by the pheromone-stimulated mitogen-activated protein (MAP) kinase Fus3 has been thought to enhance the binding of Far1 to G1-specific cyclin-dependent kinase (Cdk) complexes, thereby inhibiting their catalytic activity. Cdk-dependent phosphorylation events were invoked to account for the high instability of Far1 outside early G1 phase. To confirm any functional role of Far1 phosphorylation, we undertook a systematic mutational analysis of potential MAP kinase and Cdk recognition motifs. Two putative phosphorylation sites that strongly affect Far1 behavior were identified. A change of serine 87 to alanine prevents the cell cycle-dependent degradation of Far1, causing enhanced sensitivity to pheromone. In contrast, threonine 306 seems to be an important recipient of an activating modification, as substitutions at this position abolish the G1 arrest function of Far1. Only the phosphorylated wild-type Far1 protein, not the T306-to-A substitution product, can be found in stable association with the Cdc28-Cln2 complex. Surprisingly, Far1-associated Cdc28-Cln2 complexes are at best moderately inhibited in immunoprecipitation kinase assays, suggesting unconventional inhibitory mechanisms of Far1.

Effect of human growth hormone and insulin on [3H]thymidine incorporation, cell cycle progression, and cyclin D expression in 3T3-F442A preadipose cells.

Cellular growth- and cyclin D expression-regulating activities of GH and insulin were investigated in 3T3-F442A preadipose cells under serum-free culture conditions. The present report provides evidence that the proliferative potential of 3T3-F442A cells is reduced by GH in a time- and concentration-dependent manner based on [3H]thymidine incorporation assay and cell cycle analysis. In contrast, treatment of 3T3-F442A cells with insulin resulted in cellular proliferation. The insulin-induced proliferation of 3T3-F442A cells was diminished in the presence of GH. In an effort to define biochemical events relevant to the regulatory activities of GH and insulin on the proliferation of 3T3-F442A cells, the effects of these peptides on the expression of cyclin D were studied using Western blotting. Treatment of 3T3-F442A cells with insulin led to an increase in cyclin D expression relative to that in untreated cells. The insulin-elicited expression of cyclin D was time and dose dependent. In addition, the ability of insulin to induce cyclin D expression was reduced by GH. Our experimental results indicate that proliferation of 3T3-F442A cells was regulated by GH and insulin. The regulatory effects of GH and insulin are mediated at least in part by the alternating expression of cyclin D protein.

The regulation of Clb5 kinase activity by mating factor.

Mating factor was found to affect Clb5 kinase activity in Saccharomyces cerevisiae. Mating factor decreased Clb5 kinase activity in a time- and dose-dependent manner. The regulation of Clb5 kinase activity requires functional CLNs (G1 cyclins). Strains without functional CLNs still showed sensitivity to mating factor in the presence of moderately expressing Clb5. This type of mating factor sensitivity is thought to be induced by non-G1 arrest. It is apparent that mating factor treated cells contained inhibitor(s) of Clb5 kinase activity, suggesting that inhibition of Clb5 kinase activity is accompanied by a specific inhibitor. This notion is supported by mixing experiment. Nocodazole treatment showed that the effect of mating factor on Clb5 kinase activity occurred at G1 and connected to mitotic exit. Mating factor regulation of Clb5 kinase activity was found to be dependent on Sic1 protein.

Induction of tumor suppressor p21 protein by kinase inhibitors in MCF-7 cells.

The expression level of tumor suppressor p21 protein in response to protein kinase inhibitors was examined in MCF-7 cells. Both H7 (serine/threonine kinase inhibitor) and staurosporine (protein kinase C inhibitor) were able to induce p21 protein in a time- and dose-dependent manner. Induction of p21 by H7 but not staurosporine required the induction of p53 protein. Induction of p21 was preceded by the induction of p53 protein. Based on FACS analysis, both H7 and staurosporine act as antimitogenic agents.

Effects of tumor necrosis factor-alpha on antimitogenicity and cell cycle-related proteins in MCF-7 cells.

Tumor necrosis factor-alpha (TNF-alpha) demonstrated antimitogenic activity in MCF-7 cells (estrogen receptor-positive human breast cancer cells) in a dose- and time-dependent manner (EC-50 of 2.5 ng/ml). This antimitogenic effect of TNF-alpha was accompanied by a decreased number of cells in S phase in a dose- and time-dependent manner. Based on growth arrest experiments using aphidicolin, it is apparent that TNF-alpha acted in early G1 phase. It did not show antimitogenic effects once cells reentered the S phase based on [3H]thymidine incorporation into DNA and cell cycle analysis. Specificity of TNF-alpha was established by using monoclonal anti-human TNF-alpha antibody. On the basis of Western immunoblot analysis of Rb, p53 and cell cycle inhibitory protein (Cip1) (p21) proteins, TNF-alpha decreased Rb protein expression in a dose- and time-dependent manner whereas it increased the expression level of tumor suppressor p53 protein. TNF-alpha also increased the expression level of Cip1 (p21) protein in a dose-dependent manner. This induction of Cip1 (p21) protein was preceded by the induction of p53 protein in MCF-7 cells. Cip1 (p21) protein associated with cyclin D was also increased. Tumor suppressor Rb protein expression was increased during G1 to S phase progression. Cyclin D protein expression levels were not changed in response to TNF-alpha treatment, although serine/threonine kinase inhibitors such as H7 and the protein kinase C inhibitor staurosporine decreased cyclin D expression levels in MCF-7 cells. Based on experiments with staurosporine, it appears that TNF-alpha does not utilize a protein kinase C pathway in MCF-7 cells. Other cell cycle-related proteins such as Cdk2, Cdc2, and Cdk4 did not show any change in response to TNF-alpha. TNF-alpha did not affect complexes between cyclin D and Cdk2, Cdk4, and Rb proteins in MCF-7 cells. Taken together these results suggest that Rb, p53, and Cip1 (p21) proteins mediate TNF-alpha antimitogenic activity, and TNF-alpha induces growth arrest in the G1 phase in MCF-7 cells.

Mitogenic response to TGF-beta in 3T3-F442A cells.

In 3T3-F442A cells, TGF-beta caused cellular proliferation in a time and dose-dependent manner. TGF-beta induced cyclin D1 and cdk2 proteins in 3T3-F442A cells. The mitogenic effect of TGF-beta was specific in nature. The antimitogenic agent, hGH, inhibited the mitogenic effect of TGF-beta and was associated with inhibition of cyclin D1 expression. The protein kinase c inhibitor, staurosporine, inhibited the mitogenic effect of TGF-beta. Taken together, these results suggest that TGF-beta affects expression levels of cell cycle-regulated proteins and its mitogenic effect is mediated through protein kinase C in 3T3-F442A cells.

Cyclin-specific START events and the G1-phase specificity of arrest by mating factor in budding yeast.

The START cell cycle transition in the budding yeast Saccharomyces cerevisiae is catalyzed by the Cdc28 cyclin-dependent kinase associated with Cln-type cyclins. Since ectopic expression of the B-type cyclin CLB5 can efficiently rescue the inviability that results from CLN depletion, we tested the specificity of the CLN and CLB classes of cyclins for promoting START-associated events. Several aspects of the regulation of the mating factor response were compared for cells in which START activity was provided by either Cln-cyclins or Clb5. Unlike Cln1 and Cln2, high level expression of Clb5 was unable to repress the activity of the mating factor response pathway at START. Downregulation of Far1 protein at START is normal in cln- GAL1::CLB5 cells. Even though the Clb5-Cdc28 kinase activity in cln- GAL1::CLB5 cells is not downregulated in response to mating factor, cells arrest in the first cycle after addition of mating factor with a similar sensitivity as wild-type cells. However, whereas wild-type cells treated with mating factor arrest specifically in G1 phase as unbudded cells with unreplicated DNA (pre-START), most cln- GAL1::CLB5 cells arrest as budded post-START cells with replicated DNA. Our findings demonstrate the ability of post-START cells to arrest in response to mating factor and provide novel evidence for mechanisms that contribute to restrict mating factor-induced arrest in wild-type cells to the G1 phase of the cell cycle.

Mitogenic and receptor activities of human growth hormone 108-129.

We have selectively synthesized a number of peptides encompassing the region of helix 3 of growth hormone (GH). These peptides and native human (h) GH have been evaluated for mitogenic and receptor activities in 3T3-F442A preadipocytes. In this system, wild type hGH is anti-mitogenic. In contrast, hGH 108-129 stimulated DNA synthesis while other GH-derived peptides were ineffective. hGH (L) 108-129 had an EC50 of about 0.2 nM and was maximally effective at about 0.5 nM in stimulating [3H]thymidine incorporation in 3T3-F442A cells. hGH (L) 108-129 was mitogenically as active as insulin-like growth factor-I and more active than insulin. It was less effective than transforming growth factor-beta. By cell cycle analysis, hGH (L) 108-129 increased the proportion of cells in S/G2/M phases to 28%. hGH, when coincubated with hGH (L) 108-129, blocked the mitogenic response of the peptide. A monoclonal antibody to the GH receptor significantly reduced binding of 125I-hGH to its receptor but had no effect on binding of 125I-hGH (L) 108-129. Affinity cross-linking of 125I-hGH to its receptor was not duplicated with 125I-hGH (L) 108-129. No other GH peptides or insulin competed for binding of 125I-hGH 108-129. Scatchard analysis indicated a Kd of 5.2 nM with 5.6 x 10(5) binding sites/cell for hGH (L) 108-129. These studies indicate that hGH (L) 108-129, a sequence encompassing helix 3 of hGH, acts by binding to a site other than the GH receptor and evokes high mitogenic responses.

Human major HSP70 protein complements the localization and functional defects of cytoplasmic mutant SV40 T antigen in Swiss 3T3 mouse fibroblast cells.

The CT3 cytoplasmic localization mutant of SV40 T antigen is neither properly transported to the nucleus nor is it functional in rodent cells. Human precrisis cells are able to complement this mutation, as they are fully transformed by CT3 with wild-type efficiency. The human-specific factors responsible for this species-specific difference in response to CT3 were localized to human chromosome 6 by synteny in a panel of six somatic cell hybrids. A major human HSP70 heat shock protein located on chromosome 6 is expressed constitutively in human cells. Hsp70 proteins have been reported to play a role in intracellular movement of newly synthesized proteins. To test whether human HSP70 played a role in the complementation by human cells of the defect of CT3, we constructed a series of mouse cell lines expressing human HSP70 and tested them for their ability to localize CT3 T antigen in the nucleus and for their ability to be transformed by CT3 DNA. Mouse cell lines expressing human HSP70 protein were able to translocate mutant CT3 T antigen into the nucleus and were transformed by CT3 at rates comparable with wild-type SV40. Mouse-inducible HSP70 protein was not able to translocate cytoplasmic T antigen in Swiss 3T3 mouse fibroblast cells, even after heat shock. Apparently human HSP70 is capable of complementing directly or indirectly the structural and functional alterations in SV40 T antigen introduced by the CT3 mutation.

Three novel mutations of the PKD1 gene in Korean patients with autosomal dominant polycystic kidney disease.

Mutations at the PKD1 locus account for 85% of cases of the common genetic disorder called autosomal dominant polycystic kidney disease (ADPKD). Screening for mutations of the PKD1 gene is complicated by the genomic structure of the 5'-duplicated region encoding 75% of the gene. To date, more than 90 mutations of the PKD1 gene have been reported in the European and American populations, and relatively little information is available concerning the pattern of mutations present in the Asian populations. We looked for mutations of the PKD1 gene in 51 unrelated Korean ADPKD patients, using polymerase chain reaction (PCR) with primer pairs located in the 3' single-copy region of the PKD1 gene and by single-strand conformation polymorphism (SSCP) analysis. We found three novel mutations, a G to A substitution at nucleotide 11012 (G3601S), a C to A substitution at nucleotide 11312 (Q3701X), and a C to T substitution at nucleotide 12971 (P4254S), and a single polymorphism involving a G to C substitution at nucleotide 11470 (L3753L). These mutations were not found in control individuals, and no other mutations in the 3' single-copy region of the PKD1 gene of patients with these mutations were observed. In particular, P4254S segregated with the disease phenotype. The clinical data of affected individuals from this study, and of previously reported Korean PKD1 mutations, showed that patients with frameshift or nonsense mutations were more prone to develop end-stage renal failure than those with missense mutations. Our findings indicate that many different PKD1 mutations are likely to be responsible for ADPKD in the Korean population, as in the Western population.