p27 cytoplasmic localization is regulated by phosphorylation on Ser10 and is not a prerequisite for its proteolysis.

The activity of the cyclin-dependent kinase inhibitor p27 is controlled by its concentration and subcellular localization. However, the mechanisms that regulate its intracellular transport are poorly understood. Here we show that p27 is phosphorylated on Ser10 in vivo and that mutation of Ser10 to Ala inhibits p27 cytoplasmic relocalization in response to mitogenic stimulation. In contrast, a fraction of wild-type p27 and a p27(S10D)-phospho-mimetic mutant translocates to the cytoplasm in the presence of mitogens. G1 nuclear export of p27 and its Ser10 phosphorylation precede cyclin-dependent kinase 2 (Cdk2) activation and degradation of the bulk of p27. Interestingly, leptomycin B-mediated nuclear accumulation accelerates the turnover of endogenous p27; the p27(S10A) mutant, which is trapped in the nucleus, has a shorter half-life than wild-type p27 and the p27(S10D) mutant. In summary, p27 is efficiently degraded in the nucleus and phosphorylation of Ser10 is necessary for the nuclear to cytoplasmic redistribution of a fraction of p27 in response to mitogenic stimulation. This cytoplasmic localization may serve to decrease the abundance of p27 in the nucleus below a certain threshold required for activation of cyclin-Cdk2 complexes.

Human Cdc25 A inactivation in response to S phase inhibition and its role in preventing premature mitosis.

The Cdc25 A phosphatase is required for the G1-S transition of the cell cycle and is overexpressed in human cancers. We found that it is ubiquitylated and rapidly degraded by the proteasome and that its levels increase from G1 until mitosis. By treating cells with the DNA synthesis inhibitor hydroxyurea, Cdc25 A rapidly decreased in abundance, and this was accompanied by an increase in Cdk2 phosphotyrosine content and a decrease in Cdk2 kinase activity. Cdc25 A overexpression altered the ability of cells to arrest in the presence of hydroxyurea, and caused them to undergo premature chromosome condensation. Cdc25 A overexpression could render tumor cells less sensitive to DNA replication checkpoints, thereby contributing to their genomic instability.

Proteasome-dependent degradation of p27/kip1 in gliomas.

p27/kip1 regulates the G1-S transition of the cell cycle by inhibiting cyclin D-CDK4, cyclin E-CDK2, and cyclin A-CDK2. Modulation of p27 cellular abundance occurs mainly at post-translational level by the ubiquitin-proteasome proteolysis. Although rearrangements and mutations of p27/kip1 are extremely rare events, p27 levels are reduced and associated with a poor prognosis in many human carcinomas. In astrocytic tumors, p27 decreases with advancing anaplasia and is almost absent in glioblastomas. To verify whether the degradation of p27 protein was responsible for its reduced levels in malignant gliomas, p27 degradation activity was tested in 22 tissue extracts that represented high, low, and absent p27 protein levels. p27 protein expression was detected by immunohistochemistry and immunoblot analysis and comparable results between the 2 methods were obtained. Low or undetectable p27 degradation activity was found in samples that displayed high levels of p27, i.e. all 4 normal brain biopsies, and 4 out of 6 grade II astrocytomas. Enhanced degradation activity resulted in malignant gliomas with low or absent p27 protein levels. The proteasome inhibitor LLnL abolished p27 degradation, demonstrating that it occurs in a proteasome-dependent manner. These data suggest that proteasome degradation of p27 may be instrumental in the deregulation of the cell cycle and to the malignant transformation of gliomas.

Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation.

The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by the ubiquitin-proteasome system. Activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/Cdk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether threonine 187 phosphorylation stimulates p27 degradation through the ubiquitin-proteasome system or an alternative pathway is still not known. Here, we demonstrate that p27 ubiquitination (as assayed in vivo and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G1-enriched extracts only upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, we show that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G1 extracts. Furthermore, another p27 mutant [p27(CK-)] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor.

UBPY: a growth-regulated human ubiquitin isopeptidase.

The ubiquitin pathway has been implicated in the regulation of the abundance of proteins that control cell growth and proliferation. We have identified and characterized a novel human ubiquitin isopeptidase, UBPY, which both as a recombinant protein and upon immunoprecipitation from cell extracts is able to cleave linear or isopeptide-linked ubiquitin chains. UBPY accumulates upon growth stimulation of starved human fibroblasts, and its levels decrease in response to growth arrest induced by cell-cell contact. Inhibition of UBPY accumulation by antisense plasmid microinjection prevents fibroblasts from entering S-phase in response to serum stimulation. By increasing or decreasing the cellular abundance of UBPY or by overexpressing a catalytic site mutant, we detect substantial changes in the total pattern of protein ubiquitination, which correlate stringently with cell proliferation. Our results suggest that UBPY plays a role in regulating the overall function of the ubiquitin-proteasome pathway. Affecting the function of a specific UBP in vivo could provide novel tools for controlling mammalian cell proliferation.

11th European Cell Cycle Conference: April 23-26, 1997, Gardone Riviera (Italy) and Symposium on Cell Cycle Regulation (17th International Congress of Biochemistry and Molecular Biology): August 24-29, 1997, San Francisco, CA.

In the following pages I have summarized some of the findings presented at two recent 'cell cycle gatherings'. I have focused on those topics which in my opinion represent a substantial advancement in our understanding of the cell cycle regulatory pathways.

Interaction between Cdc37 and Cdk4 in human cells.

Using the yeast two-hybrid system we have identified novel potential Cdk4 interacting proteins. Here we described the interaction of Cdk4 with a human homologue of the yeast Drosophila CDC37 gene products. Cdc37 protein specifically interacts with Cdk4 and Cdk6, but not with Cdc2, Cdk2, Cdk3, Cdk5 and any of a number of cyclins tested. Cdc37 is not an inhibitor nor an activator of the Cdk4/cyclin D1 kinase, while it appears to facilitate complex assembly between Cdk4, and cyclin D1 in vitro. Cdc37 competes with p16 for binding to Cdk4, suggesting that p16 might exert part of its inhibitory function by affecting the formation of Cdk4/cyclin D1 complexes via Cdc37.

Increased proteasome-dependent degradation of the cyclin-dependent kinase inhibitor p27 in aggressive colorectal carcinomas.

The cell-cycle inhibitor p27 is a potential tumor suppressor, but its gene has never been found inactivated in human tumors. Because cell-cycle regulation of p27 cellular abundance occurs at the post-transcriptional level, we analyzed p27 protein expression and degradation in human colorectal carcinomas. Proteasome-mediated degradation activity of p27 was compared with its protein levels in a subset of tumor samples. We found that carcinomas with low or absent p27 protein displayed enhanced proteolytic activity specific for p27, suggesting that low p27 expression can result from increased proteasome-mediated degradation rather than altered gene expression. Patients whose tumors expressed p27 had a median survival of 151 months, whereas patients who lacked p27 (10%) had a median survival of 69 months. By multivariate analysis, p27 was found to be an independent prognostic marker. Lack of p27 was associated with poor prognosis (2.9 risk ratio for death; P = 0.003). The absence of p27 protein expression is thus a powerful negative prognostic marker in colorectal carcinomas, particularly in stage II tumors, and thereby may help in the selection of patients who will benefit from adjuvant therapy. These data suggest that aggressive tumors may result from the selection of a clone or clones that lack p27 due to increased proteasome-mediated degradation.

Cell cycle: will the real Cdk-activating kinase please stand up.

Activation of a cyclin-dependent kinase (Cdk) requires its association with a cyclin subunit and phosphorylation on a conserved residue by the Cdk-activating kinase (CAK). The discovery in budding yeast of a novel CAK that is structurally distinct from the CAKs of other eukaryotes raises questions about the 'true' CAK.

Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27.

The p27 mammalian cell cycle protein is an inhibitor of cyclin-dependent kinases. Both in vivo and in vitro, p27 was found to be degraded by the ubiquitin-proteasome pathway. The human ubiquitin-conjugating enzymes Ubc2 and Ubc3 were specifically involved in the ubiquitination of p27. Compared with proliferating cells, quiescent cells exhibited a smaller amount of p27 ubiquitinating activity, which accounted for the marked increase of p27 half-life measured in these cells. Thus, the abundance of p27 in cells is regulated by degradation. The specific proteolysis of p27 may represent a mechanism for regulating the activity of cyclin-dependent kinases.

Mammalian G1 cyclins.

D- and E-type cyclins regulate the progression of mammalian cells through the G1 phase of the cell cycle. The mechanisms responsible for the accumulation and activation of kinases dependent on cyclins D and E in both normal and cancerous cells have recently been uncovered. Overexpression of cyclin D1 protein as a consequence of genetic rearrangements, and deletions or mutations of the p16INK4 gene have been demonstrated in a large variety of human cancers, including breast and esophageal carcinomas, lymphomas, bladder carcinoma, pancreatic adenocarcinoma and familial melanoma.

Differential expression and regulation of Cyclin D1 protein in normal and tumor human cells: association with Cdk4 is required for Cyclin D1 function in G1 progression.

In this study we have surveyed by immunoblotting the protein levels of Cyclin D1, D2, D3 and their catalytic partners, Cdk4 and Cdk6 in normal and transformed human cells. We found that all these proteins were differentially expressed in diploid cells derived from different tissues, in contrast to Cyclin E, Cyclin A and Cdk2 which are ubiquitously expressed. D-type Cyclins were never dramatically overexpressed and often very poorly expressed in tumor cell lines when compared to the levels in their normal counterparts. In contrast, Cdk4 was expressed at high levels in several tumor cell lines and Cdk6 was ectopically expressed in two sarcoma lines, suggesting a possible involvement of these two Cdks in oncogenesis. Interestingly, low levels of Cyclin D1 and D3 proteins always correlated with functional inactivation of the retinoblastoma gene product (pRb). In cells displaying active pRb, Cyclin D1 was found associated with Cdk4 regardless of whether the p53 gene was wild-type or mutant. Microinjection during G1 of Cyclin D1 anti-sense cDNA or anti-Cyclin D1 antibody in these cells arrested the cell cycle in G1. In cells lacking pRb function, Cyclin D1 was dissociated from Cdk4. Microinjection during G1 of Cyclin D1 antisense cDNA or anti-Cyclin D1 antibody in these cells did not affect G1 progression. These results show that (i) in the absence of pRb, Cyclin D1 is expressed at low levels, is dissociated from Cdk4 and becomes dispensable in G1; (ii) Cyclin D1 needs to be associated with its catalytic subunit, Cdk4, to function as a positive regulator of G1 progression.

Cyclin D1-mediated inhibition of repair and replicative DNA synthesis in human fibroblasts.

Cyclin D1 is a key regulator of the G1 phase of the cell cycle. Inhibition of cyclin D1 function results in cell cycle arrest, whereas unregulated expression of the protein accelerates G1. Cyclin D1 is localized to the nucleus during G1. We found that during repair DNA synthesis, subsequent to UV-induced DNA damage, G1 cells readily lost their cyclin D1 while the proliferating cell nuclear antigen (PCNA) tightly associated with nuclear structures. Microinjection of cyclin D1 antisense accelerated DNA repair, whereas overexpression of cyclin D1 prevented DNA repair and the relocation of PCNA after DNA damage. Coexpression of cyclin D1 with its primary catalytic subunit, Cdk4, or with Cdk2, also prevented repair. In contrast, coexpression of PCNA, which is also a cyclin D1-associated protein, restored the ability of cells to repair their DNA. Acute overexpression of cyclin D1 in fibroblasts prevented them from entering S phase. Again, these effects were abolished by coexpression of cyclin D1 together with PCNA, but not with Cdk4 or Cdk2. Altogether, these results indicate that down-regulation of cyclin D1 is necessary for PCNA relocation and repair DNA synthesis as well as for the start of DNA replication. Cyclin D1 appears to be an essential component of a G1-checkpoint.

The phosphorylation of calmodulin and calmodulin fragments by kinase fractions from bovine brain.

The phosphorylation of intact calmodulin and of fragments obtained by trypsin digestion was studied, using a protein kinase partially purified from bovine brain. Brain extracts were made in the presence of the detergent CHAPS (3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate). The protein kinase catalyzed the incorporation of nearly 1 mol of 32P from [gamma-32P]ATP into calmodulin fragment 1-106. Incorporation was exclusively into serine 101. With fragment 78-148, the extent of phosphorylation was somewhat less and 32P appeared mainly in threonine residues. Fragment 1-90 was also a fairly good substrate, but the phosphorylation of intact calmodulin never exceeded 0.01 mol per mol. Little or no phosphorylation was seen with parvalbumin, the brain Ca2+-binding protein (CBP-18) and intestinal calcium-binding protein. The protein kinase had no requirement for cAMP or phospholipids. High levels of Mg2+ (60-70 mM) stimulated phosphorylation of the fragments 20-fold. Millimolar concentrations of Ca2+ were inhibitory. It is suggested that the calmodulin fragments were in a conformation more favorable for phosphorylation than intact soluble calmodulin.

Genetically engineered calmodulins differentially activate target enzymes.

Three mutant calmodulin (CaM) genes together with the normal chicken CaM cDNA have been expressed in bacteria for the purpose of determining structure/function relationships in CaM. The mutant CaM genes were generated by in vitro recombination between a chicken CaM cDNA and a processed pseudogene that encodes a full-length CaM but with 19 amino acid substitutions as compared to authentic vertebrate CaM. The calmodulin-like (CaML) proteins derived from the pseudogene are called CaML19, CaML16, and CaML3 and contain 19, 16, and 3 amino acid substitutions, respectively. CaML3 is functionally identical to CaM by all criteria tested. The functional characteristics of CaML16 and CaML19 are also indistinguishable yet quite different from normal CaM. CaML19 and CaML16 will maximally activate myosin light chain kinase but will only half-maximally activate calcineurin and CaM-dependent multiprotein kinase. In addition, CaML16 and CaML19 do not activate phosphorylase kinase. The differential activation of these enzymes does not result from the loss of Ca2+-binding sites, since CaML16 binds four Ca2+ with affinity similar to CaM or CaM23. It is more likely that the functional characteristics of the mutant proteins result from an altered tertiary structure, since the Ca2+-dependent enhancement of tyrosine fluorescence and limited proteolysis pattern of CaML16 are different from that of CaM. The data demonstrate that the nature of the interaction of CaM with myosin light chain kinase is different from its interaction with calcineurin, CaM-dependent multiprotein kinase, and phosphorylase kinase and may involve different functional domains in CaM.

Inhibition of adenylate cyclase by transglutaminase-catalyzed reactions in pigeon erythrocyte ghosts.

We report the occurrence in pigeon erythrocytes of a soluble Ca2+-dependent transglutaminase (TGase) activity. The effect of the erythrocyte ghost protein modifications, determined by TGase-catalyzed reactions, on adenylate cyclase, phospholipid methyltransferase I and II activities and on the lipidic matrix fluidity of the membrane was investigated by using a purified guinea pig liver TGase preparation. The results showed a significant inhibitory effect of such modifications both on the basal and on the variously stimulated (by NaF, Gpp(NH)p alone or in the presence of 1-isoproterenol) adenylate cyclase activity. By contrast, both the phospholipid methylation and the fluidity of the lipidic matrix of the membrane were unaffected by TGase-mediated reactions. These data suggest a new possible inhibitory mechanism of the cyclic AMP synthesis which might be triggered by the enhancement of the cytosolic Ca2+ concentration.

Do porins inhibit the macrophage phagocyting activity by stimulating the adenylate cyclase?

Porins interact with macrophage membranes and inhibit their phagocyting activity. We have tested the porin effect on a biologically relevant membrane-bound enzymic activity, the adenylate cyclase system, which appears to be stimulated both in the presence of Mn2+ and Mg2+ or Mg2+ + Gpp(NH)p. Moreover, for mice macrophages incubated in the presence of porins, there is an increase in the intracellular cAMP content after 5 min of incubation, with a maximum after 15 min of incubation. The results shown suggest that the porin effects on the adenylate cyclase can represent the molecular basis of the porin-dependent inhibition of the macrophages phagocytosis. Our point of view, which proposes a cAMP role in inhibiting the phagocyting activity in macrophages, is supported also by the results of the experiments carried out in the presence of both dibutyryl-cAMP or aminophylline. The phagocyting activity is inhibited in all cases and independently of the bacteria to be phagocyted.

The effects of lidocaine and procainamide on the sarcolemmal membrane high affinity cyclic AMP phosphodiesterase of rat myocardium.

The incubation of rat myocardial sarcolemmal membranes with 10(-4)M lidocaine or procainamide results in a decreased fluorescence polarization of the extrinsic probes (diphenylhexatriene and 12-anthroylstearate) and in the stimulation of the membrane-bound high affinity cyclic AMP phosphodiesterase activity (PDE). Lidocaine or procainamide do not contrast, but facilitate the aminophylline inhibitory action on the PDE activity, without modifying the basic molecular mechanism of the inhibitory action. The amplitudes of the effects described are inversely correlated to the temperature, being greater at the lower tested temperature (25 degrees greater than 30 degrees greater than 37 degrees C).