D-type cyclin-dependent kinase activity in mammalian cells.

D-type cyclin-dependent kinase activities have not so far been detected in mammalian cells. Lysis of rodent fibroblasts, mouse macrophages, or myeloid cells with Tween 20 followed by precipitation with antibodies to cyclins D1, D2, and D3 or to their major catalytic partner, cyclin-dependent kinase 4 (cdk4), yielded kinase activities in immune complexes which readily phosphorylated the retinoblastoma protein (pRb) but not histone H1 or casein. Virtually all cyclin D1-dependent kinase activity in proliferating macrophages and fibroblasts could be attributed to cdk4. When quiescent cells were stimulated by growth factors to enter the cell cycle, cyclin D1-dependent kinase activity was first detected in mid G1, reached a maximum near the G1/S transition, and remained elevated in proliferating cells. The rate of appearance of kinase activity during G1 phase lagged significantly behind cyclin induction and correlated with the more delayed accumulation of cdk4 and formation of cyclin D1-cdk4 complexes. Thus, cyclin D1-associated kinase activity was not detected during the G0-to-G1 transition, which occurs within the first few hours following growth factor stimulation. Rodent fibroblasts engineered to constitutively overexpress either cyclin D1 alone or cyclin D3 together with cdk4 exhibited greatly elevated cyclin D-dependent kinase activity, which remained absent in quiescent cells but rose to supraphysiologic levels as cells progressed through G1. Therefore, despite continued enforced overproduction of cyclins and cdk4, the assembly of cyclin D-cdk4 complexes and the appearance of their kinase activities remained dependent upon serum stimulation, indicating that upstream regulators must govern formation of the active enzymes.     

A- and B-type cyclins differentially modulate substrate specificity of cyclin-cdk complexes.

Both cyclins A and B associate with and thereby activate cyclin-dependent protein kinases (cdks). We have investigated which component in the cyclin-cdk complex determines its substrate specificity. The A- and B-type cyclin-cdk complexes phosphorylated histone H1 and their cyclin subunits in an indistinguishable manner, irrespective of the catalytic subunit, p33cdk2 or p34cdc2. In contrast, only the cyclin A-cdk complexes phosphorylated the Rb-related p107 protein in vitro. Likewise, binding studies revealed that cyclin A-cdk complexes bound stably to p107 in vitro, whereas cyclin B-cdk complexes did not detectably associate with p107, under identical assay conditions. Binding to p107 required both cyclin A and a cdk as neither subunit alone bound to p107. These results demonstrate that although the kinase subunit provides a necessary component for binding, it is the cyclin subunit that plays the critical role in targeting the complex to p107. Finally, we show that the cyclin A-p33cdk2 complex phosphorylated p107 in vitro at most of its sites that are also phosphorylated in human cells, suggesting that the cyclin A-p33cdk2 complex is a major kinase for p107 in vivo.     

Transforming activity of Fbxo7 is mediated specifically through regulation of cyclin D/cdk6

D cyclins (D1, D2 and D3) and their catalytic subunits (cyclin-dependent kinases cdk4 and cdk6) have a facilitating, but nonessential, role in cell cycle entry. Tissue-specific functions for D-type cyclins and cdks have been reported; however, the biochemical properties of these kinases are indistinguishable. We report that an F box protein, Fbxo7, interacted with cellular and viral D cyclins and distinguished among the cdks that bind D-type cyclins, specifically binding cdk6, in vitro and in vivo. Fbxo7 specifically regulated D cyclin/cdk6 complexes: Fbxo7 knockdown decreased cdk6 association with cyclin and its overexpression increased D cyclin/cdk6 activity and E2F activity. Fbxo7 interacted with p27, but its enhancement of cyclin D/cdk6 activity was p21/p27 independent. Fbxo7 overexpression transformed murine fibroblasts, rendering them tumorigenic in athymic nude mice. Transformed phenotypes were dependent on cdk6, as knockdown of cdk6 reversed them. Fbxo7 was highly expressed in epithelial tumors, but not in normal tissues, suggesting that it may have a proto-oncogenic role in human cancers.     

Activation of cyclin-dependent kinase 4 (cdk4) by mouse MO15-associated kinase.

The assembly of functional holoenzymes composed of regulatory D-type cyclins and cyclin-dependent kinases (cdks) is rate limiting for progression through the G1 phase of the mammalian somatic cell cycle. Complexes between D-type cyclins and their major catalytic subunit, cdk4, are catalytically inactive until cyclin-bound cdk4 undergoes phosphorylation on a single threonyl residue (Thr-172). This step is catalyzed by a cdk-activating kinase (CAK) functionally analogous to the enzyme which phosphorylates cdc2 and cdk2 at Thr-161/160. Here, we demonstrate that the catalytic subunit of mouse cdc2/cdk2 CAK (a 39-kDa protein designated p39MO15) can assemble with a regulatory protein present in either insect or mammalian cells to generate a CAK activity capable of phosphorylating and enzymatically activating both cdk2 and cdk4 in complexes with their respective cyclin partners. A newly identified 37-kDa cyclin-like protein (cyclin H [R. P. Fisher and D. O. Morgan, Cell 78:713-724, 1994]) can assemble with p39MO15 to activate both cyclin A-cdk2 and cyclin D-cdk4 in vitro, implying that CAK is structurally reminiscent of cyclin-cdk complexes themselves. Antisera produced to the p39MO15 subunit can completely deplete mammalian cell lysates of CAK activity for both cyclin A-cdk2 and cyclin D-cdk4, with recovery of activity in the resulting immune complexes. By using an immune complex CAK assay, CAK activity for cyclin A-cdk2 and cyclin D-cdk4 was detected both in quiescent cells and invariantly throughout the cell cycle. Therefore, although it is essential for the enzymatic activation of cyclin-cdk complexes, CAK appears to be neither rate limiting for the emergence of cells from quiescence nor subject to upstream regulatory control by stimulatory mitogens.     

Low levels of cyclin D and nonfunctional Rb protein affect cdk6 association with cyclin-dependent kinase inhibitor p27(Kip1)

p27(Kip1) associates with cyclin/cdk complexes and inhibiting cdk activity, and overexpression of p27(Kip1) induces G1 arrest. We found that p27(Kip1) overexpression inhibits cdk2 kinase activity, but not cdk6 kinase activity in HeLa cells. The amount of p27(Kip1) associated with cdk2 was significantly higher than that associated with cdk6. cdk6 complexes contained detectable amounts of p27(Kip1) in all human cell lines examined, except in HeLa cells where p27(Kip1) preferentially associated with cdk2. It appears that in HeLa cells overexpressed p27(Kip1) fails to inhibit cdk6 kinase activity because of low binding affinity of cdk6 to p27(Kip1). The low binding affinity is due to a low level of the cdk6/cyclin D complexes. Functional inactivation of pRb has an effect on p27(Kip1) association with cdk6/cyclin D complexes.     

Members of the NAP/SET family of proteins interact specifically with B- type cyclins

Cyclin-dependent kinase complexes that contain the same catalytic subunit are able to induce different events at different times during the cell cycle, but the mechanisms by which they do so remain largely unknown. To address this problem, we have used affinity chromatography to identify proteins that bind specifically to mitotic cyclins, with the goal of finding proteins that interact with mitotic cyclins to carry out the events of mitosis. This approach has led to the identification of a 60-kD protein called NAP1 that interacts specifically with members of the cyclin B family. This interaction has been highly conserved during evolution: NAP1 in the Xenopus embryo interacts with cyclins B1 and B2, but not with cyclin A, and the S. cerevisiae homolog of NAP1 interacts with Clb2 but not with Clb3. Genetic experiments in budding yeast indicate that NAP1 plays an important role in the function of Clb2, while biochemical experiments demonstrate that purified NAP1 can be phosphorylated by cyclin B/p34cdc2 kinase complexes, but not by cyclin A/p34cdc2 kinase complexes. These results suggest that NAP1 is a protein involved in the specific functions of cyclin B/p34cdc2 kinase complexes. In addition to NAP1, we found a 43-kD protein in Xenopus that is homologous to NAP1 and also interacts specifically with B-type cyclins. This protein is the Xenopus homolog of the human SET protein, which was previously identified as part of a putative oncogenic fusion protein (Von Lindern et al., 1992).     

Cyclin A- and cyclin B-dependent protein kinases are regulated by different mechanisms in Xenopus egg extracts.

Cyclins are proteins which are synthesized and degraded in a cell cycle-dependent fashion and form integral regulatory subunits of protein kinase complexes involved in the regulation of the cell cycle. The best known catalytic subunit of a cyclin-dependent protein kinase complex is p34cdc2. In the cell, cyclins A and B are synthesized at different stages of the cell cycle and induce protein kinase activation with different kinetics. The kinetics of activation can be reproduced and studied in extracts of Xenopus eggs to which bacterially produced cyclins are added. In this paper we report that in egg extracts, both cyclin A and cyclin B associate with and activate the same catalytic subunit, p34cdc2. In addition, cyclin A binds a less abundant p33 protein kinase related to p34cdc2, the product of the cdk2/Eg1 gene. When complexed to cyclin B, p34cdc2 is subject to transient inhibition by tyrosine phosphorylation, producing a lag between the addition of cyclin and kinase activation. In contrast, p34cdc2 is only weakly tyrosine phosphorylated when bound to cyclin A and activates rapidly. This finding shows that a given kinase catalytic subunit can be regulated in a different manner depending on the nature of the regulatory subunit to which it binds. Tyrosine phosphorylation of p34cdc2 when complexed to cyclin B provides an inhibitory check on the activation of the M phase inducing protein kinase, allowing the coupling of processes such as DNA replication to the onset of metaphase. Our results suggest that, at least in the early Xenopus embryo, cyclin A-dependent protein kinases may not be subject to this checkpoint and are regulated primarily at the level of cyclin translation.     

Multiple Functions of D-Type Cyclins Can Antagonize pRb-Mediated Suppression of Proliferation

The most well understood function of the D-type cyclins is to activate the G1kinases, cdk4 and cdk6, and target the retinoblastoma gene product (pRb) forphosphorylation and inactivation. pRb can suppress S phase entry, cause a transientG1 arrest following DNA damage, and is critical in establishing terminal cell cyclewithdrawal in cells exposed to differentiation or senescence-inducing signals. Each ofthese functions of pRb can be demonstrated in cultured cells derived from humantumors that have suffered RB1 gene inactivation. In such in vitro assays, coexpressionof D type cyclins has been shown to inhibit the function of pRb, likelyreflecting an oncogenic role of cyclin D1 in vivo. Two regions of cyclin D, the LxCxEpRb-binding motif, and the cyclin box, are thought to be critical for the proper functionof cyclin D. Here we show that the LxCxE motif is dispensable in cyclin D1 for allfunctions tested, but is required by cyclin D2. This observation suggests that there isa functional difference between cyclins D1 and D2 in pRb regulation, and arguesagainst complete functional redundancy of these D cyclins. In addition, the ability ofcyclins D1 and D2 to activate cdk partners is required for induction of pRbphosphorylation and S phase entry. However, mutant forms of cyclins D1 and D2that are incapable of activating kinase partners were still able to prevent pRb-inducedsenescence. Thus, D cyclins have both kinase-dependent and kinase-independentmechanisms of interfering with proliferation arrest and senescence.     

ik3-1/Cables is a substrate for cyclin-dependent kinase 3 (cdk 3).

p70ik3-1 (a 70-kDa protein) contains a cyclin box, and binds to p35cdk3 in vivo and in vitro [Matsuoka, M., Matsuura, Y., Semba, K. & Nishimoto, I. (2000) Biochem. Biophys. Res. Commun. 273, 442-447]. In spite of its structural similarity to cyclins, p70ik3-1 does not activate cyclin-dependent kinase 3 (cdk3)-mediated phosphorylation of pRb, histone H1, or the C-terminal domain of RNA polymerase II. Here, we report that Ser274 of p70ik3-1 is phosphorylated by cdk2 or cdk3 bound to cyclin A and to cyclin E in vitro. We also found that in COS7 cells in which cyclin E and cdk3 were ectopically overexpressed, the phosphorylation level of Ser274 in coexpressed p70ik3-1 is upregulated. We therefore conclude that p70ik3-1 is a substrate for cdk3-mediated phosphorylation.     

Cyclin D3 is dispensable for human diffuse large B-cell lymphoma survival and growth: evidence for redundancy with cyclin E

Genomic changes disrupting the expression of cyclin D3 are associated with aberrant growth of several human B-lymphoid malignancies. We demonstrate that the human diffuse large B-cell lymphoma (DLBCL), OCI-LY18 (LY18) expresses cyclin D3 but not cyclins D1 and D2. RNA interference was used to deplete cyclin D3 from LY18 cells. Surprisingly, knockdown of cyclin D3 did not inhibit pRb phosphorylation on cdk4/6- and cdk2-specific residues or measurably affect viability and proliferation. These results suggest that cyclin D3 is dispensable in LY18 cell proliferation and survival. Similar results were obtained following depletion of cyclin E. By contrast, combined knockdown of cyclins D3 and E had substantial consequences leading to G1-phase arrest and inhibition of proliferation. Whereas cell cycle distribution was not affected following individual depletion of cdk4, cdk6, or cdk2, the combined knockdown of cdk4 and cdk6 led to accumulation of LY18 cells in G1-phase of the cell cycle and inhibition of proliferation. Likewise treatment of LY18 cells with 2-Bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, a selective inhibitor of cdk4/6, led to inhibition of proliferation. Taken together, these results uncover a built-in redundancy with cyclins D3 and E for G1-S progression. Moreover these findings highlight the rationale for simultaneous disruption of cdk4/6 as a potential therapeutic cancer strategy.     

Regulation of cyclin D-dependent kinase 4 (cdk4) by cdk4-activating kinase.

The accumulation of assembled holoenzymes composed of regulatory D-type cyclins and their catalytic partner, cyclin-dependent kinase 4 (cdk4), is rate limiting for progression through the G1 phase of the cell cycle in mammalian fibroblasts. Both the synthesis and assembly of D-type cyclins and cdk4 depend upon serum stimulation, but even when both subunits are ectopically overproduced, they do not assemble into complexes in serum-deprived cells. When coexpressed from baculoviral vectors in intact Sf9 insect cells, cdk4 assembles with D-type cyclins to form active protein kinases. In contrast, recombinant D-type cyclin and cdk4 subunits produced in insect cells or in bacteria do not assemble as efficiently into functional holoenzymes when combined in vitro but can be activated in the presence of lysates obtained from proliferating mammalian cells. Assembly of cyclin D-cdk4 complexes in coinfected Sf9 cells facilitates phosphorylation of cdk4 on threonine 172 by a cdk-activating kinase (CAK). Assembly can proceed in the absence of this modification, but cdk4 mutants which cannot be phosphorylated by CAK remain catalytically inactive. Therefore, formation of the cyclin D-cdk4 complex and phosphorylation of the bound catalytic subunit are independently regulated, and in addition to the requirement for CAK activity, serum stimulation is required to promote assembly of the complexes in mammalian cells.     

Disruption of the actin cytoskeleton leads to inhibition of mitogen-induced cyclin E expression, Cdk2 phosphorylation, and nuclear accumulation of the retinoblastoma protein-related p107 protein

The actin cytoskeleton has been found to be required for mitogen-stimulated cells to passage through the cell cycle checkpoint. Here we show that selective disruption of the actin cytoskeleton by dihydrocytochalasin B (H(2)CB) blocked the mitogenic effect in normal Swiss 3T3 cells, leading to cell cycle arrest at mid to late G(1) phase. Cells treated with H(2)CB remain tightly attached to the substratum and respond to mitogen-induced MAP kinase activation. Upon cytoskeleton disruption, however, growth factors fail to induce hyperphosphorylation of the retinoblastoma protein (pRb) and the pRb-related p107. While cyclin D1 induction and cdk4-associated kinase activity are not affected, induction of cyclin E expression and activation of cyclin E-cdk2 complexes are greatly inhibited in growth-stimulated cells treated with H(2)CB. The inhibition of cyclin E expression appears to be mediated at least in part at the RNA level and the inhibition of cdk2 kinase activity is also attributed to the decrease in cdk2 phosphorylation and proper subcellular localization. The expression patterns of cdk inhibitors p21 and p27 are similar in both untreated and H(2)CB-treated cells upon serum stimulation. In addition, the changes in subcellular localization of pRb and p107 appear to be linked to their phosphorylation states and disruption of normal actin structure affects nuclear migration of p107 during G(1)-to-S progression. Taken together, our results suggest that the actin cytoskeleton-dependent G(1) arrest is linked to the cyclin-cdk pathway. We hypothesize that normal actin structure may be important for proper localization of certain G(1) regulators, consequently modulating specific cyclin and kinase expression.     

Cutting edge: differential signaling requirements for activation of assembled cyclin D3-cdk4 complexes in B-1 and B-2 lymphocyte subsets

B-1 lymphocytes represent a distinct B cell subset with unusual mitogenic responses. PMA alone promotes proliferation in B-1 cells, but not in splenic B-2 cells. Although cyclin D2-cyclin-dependent kinase 4 (cdk4) complexes mediate early retinoblastoma gene product (pRb) phosphorylation in B-1 cells, the transient nature of their accumulation cannot account for the continued increase in pRb phosphorylation, which is maximal at 24 h. We show herein that PMA promotes the accumulation of functional cyclin D3-cdk4 complexes in B-1 cells following loss of cyclin D2. PMA also induces accumulation of cyclin D3-cdk4 complexes in B-2 cells; however, these complexes do not phosphorylate pRb. Thus, PMA is sufficient to induce synthesis and assembly of cyclin D3-cdk4 complexes in B-1 and B-2 cells; however, PMA triggers cyclin D3-cdk4 activation only in B-1 cells. These results reveal a novel regulatory step that controls activation of cyclin D3-cdk4 complexes whose function segregates differentially in B cell subsets.     

Cyclic AMP-dependent phosphorylation of cyclin D3-bound CDK4 determines the passage through the cell cycle restriction point in thyroid epithelial cells

According to current concepts, the cell cycle commitment after restriction (R) point passage requires the sustained stimulation by mitogens of the synthesis of labile d-type cyclins, which associate with cyclin-dependent kinase (CDK) 4/6 to phosphorylate pRb family proteins and sequester the CDK inhibitor p27kip1. In primary cultures of dog thyroid epithelial cells, the cAMP-dependent cell cycle induced by a sustained stimulation by thyrotropin or forskolin differs from growth factor mitogenic pathways, as cAMP does not upregulate d-type cyclins but increases p27 levels. Instead, cAMP induces the assembly of required cyclin D3-CDK4 complexes, which associate with nuclear p27. In this study, the arrest of forskolin stimulation rapidly slowed down the entry of dog thyrocytes into S phase and the phosphorylation of pRb family proteins. The pRb kinase activity, but not the formation, of the cyclin D3-CDK4-p27 complex was strongly reduced. Using two-dimensional gel electrophoresis, a phosphorylated form of CDK4 was separated. It appeared in response to forskolin and was bound to both cyclin D3 and p27, presumably reflecting the activating Thr-172 phosphorylation of CDK4. Upon forskolin withdrawal or after cycloheximide addition, this CDK4 phosphoform unexpectedly persisted in p27 complexes devoid of cyclin D3 but it disappeared from the more labile cyclin D3 complexes. These data demonstrate that the assembly of the cyclin D3-CDK4-p27 holoenzyme and the subsequent phosphorylation and activation of CDK4 depend on distinct cAMP actions. This provides a first example of a crucial regulation of CDK4 phosphorylation by a mitogenic cascade and a novel mechanism of cell cycle control at the R point.     

Role of cyclins in neuronal differentiation of immortalized hippocampal cells.

The proto-oncogene cyclin D1 and the neuron-specific cyclins p35 and p39 are expressed during brain maturation. To investigate the role of these cyclins in neuronal differentiation, we used a conditionally immortalized rat hippocampal cell line, H19-7, that expresses cyclin-dependent kinases 4 and 5 (cdk4 and -5). Cyclin D1, which activates cdk4 and binds but does not activate cdk5, was increased upon differentiation of the H19-7 cells. However, microinjection of either sense or antisense cyclin D1 cDNA or anti-cyclin D1 antibodies had no effect on morphological differentiation of the cells. On the other hand, neurite outgrowth was stimulated by expression of p35 or p39, both of which activate cdk5. A dominant-negative mutant of cdk5 blocked both p35- and p39-induced neurite extension as well as basic fibroblast growth factor (bFGF)-induced neuronal differentiation. However, of these cyclins, only antisense p39 prevented bFGF-induced neurite outgrowth. These studies indicate that cyclin D1 is neither necessary nor sufficient for morphological differentiation, that p35 is sufficient but not required, and that p39 is both necessary and sufficient for neurite outgrowth in the hippocampal cells. Taken together, these results represent the first demonstration of a specific role for p39 in neuronal differentiation, implicate the cyclin-activated kinase cdk5 in this process, and indicate that p39 is able to mediate neurite outgrowth in the presence or absence of cyclin D1.     

Virus-encoded cyclin.

Herpesvirus saimiri contains an open reading frame called eclf2 with homology to the cellular type D cyclins. We now show that the eclf2 gene product is a novel virus-encoded cyclin (v-cyclin). The protein encoded by the v-cyclin gene of this oncogenic herpesvirus was found to have an apparent molecular size of 29 kDa in transformed cells. v-Cyclin protein was found to be associated with cdk6, a cellular cyclin-dependent kinase known to interact with cellular type D cyclins. cdk6/v-cyclin complexes strongly phosphorylated Rb fusion protein and histone H1 as substrates in vitro. Mutational analyses showed that highly conserved amino acids in the cyclin box of v-cyclin were important for association with cdk6 and for activation of cdk6 kinase activity. Thus, v-cyclin resembles cellular type D cyclins in primary sequence, in its association with cdk6, by its ability to activate protein kinase activity, and by the presence of functional cyclin box sequences. v-Cyclin exhibited a selective preference for association with cdk6 over other cyclin-dependent kinases and a high level of kinase activation. The properties of v-cyclin suggest a likely role in oncogenic transformation by this T-lymphotropic herpesvirus.     

Distinct properties of cyclin-dependent kinase complexes containing cyclin A1 and cyclin A2

The distinct expression patterns of the two A-type cyclins during spermatogenesis and the absolute requirement for cyclin A1 in this biological process in vivo suggest that they may confer distinct biochemical properties to their CDK partners. We therefore compared human cyclin A1- and cyclin A2-containing CDK complexes in vitro by determining kinetic constants and by examining the complexes for their ability to phosphorylate pRb and p53. Differences in biochemical activity were observed in CDK2 but not CDK1 when complexed with cyclin A1 versus cyclin A2. Further, CDK1/cyclin A1 is a better kinase complex for phosphorylating potentially physiologically relevant substrates pRb and p53 than CDK2/cyclin A2. The activity of CDKs can therefore be regulated depending upon which A-type cyclin they bind and CDK1/cyclin A1 might be preferred in vivo.     

MAT1 (''menage à trois'') a new RING finger protein subunit stabilizing cyclin H-cdk7 complexes in starfish and Xenopus CAK.

The kinase responsible for Thr161-Thr160 phosphorylation and activation of cdc2/cdk2 (CAK:cdk-activating kinase) has been shown previously to comprise at least two subunits, cdk7 and cyclin H. An additional protein co-purified with CAK in starfish oocytes, but its sequencing did not reveal any similarity with any known protein. In the present work, a cDNA encoding this protein is cloned and sequenced in both starfish and Xenopus oocytes. It is shown to encode a new member of the RING finger family of proteins with a characteristic C3HC4 motif located in the N-terminal domain. We demonstrate that the RING finger protein (MAT1: 'menage à trois') is a new subunit of CAK in both vertebrate and invertebrates. However, CAK may also exist in oocytes as heterodimeric complexes between cyclin H and cdk7 only. Stable heterotrimeric CAK complexes were generated in reticulocyte lysates programmed with mRNAs encoding Xenopus cdk7, cyclin H and MAT1. In contrast, no heterodimeric cyclin H-cdk7 complex could be immunoprecipitated from reticulocyte lysates programmed with cdk7 and cyclin H mRNAs only. Stabilization of CAK complexes by MAT1 does not involve phosphorylation of Thr176, as the Thr176-->Ala mutant of Xenopus cdk7 could engage as efficiently as wild-type cdk7 in ternary complexes. Even though starfish MAT1 is almost identical to Xenopus MAT1 in the RING finger domain, the starfish subunit could not replace the Xenopus subunit and stabilize cyclin H-cdk7 in reticulocyte lysate, suggesting that the MAT1 subunit does not (or not only) interact with cyclin H-cdk7 through the RING finger domain.     

Effects of Cyclic AMP on Components of the Cell Cycle Machinery Regulating DNA Synthesis in Cultured Astrocytes

Cyclic AMP is a second messenger for various hormones that inhibits cell multiplication and DNA synthesis in cultured astrocytes. We examined the effects of increasing intracellular cyclic AMP on the catalytic (cdks) and regulatory (cyclins and ckis) components of cyclin-dependent protein kinases, which regulate progression of the cell cycle before completion of DNA synthesis, in primary cultured astrocytes and in an astrocytic cell line C.LT.T.1.1. The amount of cdk4 changed little during the cell cycle and was not affected by cyclic AMP. There was little cdk1 and cdk2 in quiescent cells, and their expression increased during the G1-S phases. Cyclic AMP strongly inhibited cdk1 and cdk2 expression. Transforming growth factor beta also inhibited cdk1 expression in primary astrocytes. Cyclic AMP did not affect the two ckis p27KIP1 and p21CIP1. There was little cyclin D1 in quiescent cells, but it increased during the G1 phase and was reduced by cyclic AMP. Cyclin E increased during the G1-S phases and was not affected by cyclic AMP in primary astrocytes. The amount of cyclin A was low in quiescent cells and increased during the G1-S phases. Expression of its mRNA and protein was inhibited by cyclic AMP. The protein kinase activities associated with complexes of cyclins and cdks were increased by growth factors and prevented by cyclic AMP. We conclude that cyclic AMP inhibits progression of the cell cycle in astrocytes at least by preventing the expression of the regulatory subunits, cyclins D1 and A, and catalytic subunits, cdk1 and cdk2, of cyclin-regulated protein kinases. Key Words: Cyclin-dependent protein kinases-Glial cells-Cyclic AMP.