Kaposi's sarcoma-associated herpesvirus encodes a functional cyclin.

Kaposi's sarcoma-associated herpesvirus (KSHV) (also called human herpesvirus 8) is consistently found in Kaposi's sarcoma lesions and in body-cavity-based lymphomas. A 17-kb KSHV lambda clone was obtained directly from a Kaposi's sarcoma lesion. DNA sequence analysis of this clone identified an open reading frame which has 32% amino acid identity and 53% similarity to the virus-encoded cyclin (v-cyclin) of herpesvirus saimiri (HVS) and 31% identity and 53% similarity to human cellular cyclin D2. This KSHV open reading frame was shown to encode a 29- to 30-kDa protein with the properties of a v-cyclin. KSHV v-cyclin protein was found to associate predominantly with cdk6, a cellular cyclin-dependent kinase known to interact with cellular type D cyclins and HVS v-cyclin. The KSHV v-cyclin was also found to associate weakly with cdk4. KSHV v-cyclin-cdk6 complexes strongly phosphorylated glutathione S-transferase-Rb fusion protein and histone H1 as substrates in vitro. Thus, KSHV v-cyclin resembles the v-cyclin of the T-lymphocyte-transforming HVS in its specificity for association with cdk6 and in its ability to strongly activate cdk6 protein kinase activity.     

The cyclin encoded by Kaposi's sarcoma-associated herpesvirus stimulates cdk6 to phosphorylate the retinoblastoma protein and histone H1.

Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) is a novel gammaherpesvirus implicated in the cause of Kaposi's sarcoma and certain malignancies of lymphatic origin. One of the candidate genes possibly involved in promoting tumor development is an open reading frame (ORF) with sequence similarity to human type D cyclin genes. This cyclin-like gene, when expressed in tissue culture cells, promotes phosphorylation and inactivation of the retinoblastoma tumor suppressor protein and thereby may result in deregulation of cell division control. We report here the biochemical characterization of this cyclin (KSHV-cyc) and the kinase activity that it elicits upon expression in tissue culture cells. We demonstrate that the kinase activity associated with KSHV-cyc is sensitive to the cdk inhibitor p27 (KIP) and due to activation of cdk6. However, in contrast to cdk6 activated by cellular type D cyclins, the cdk6 activated by KSHV-cyc is capable of phosphorylating not only the retinoblastoma protein but also histone H1. This finding implies that activation by KSHV-cyc alters the substrate preference of this cdk. This may have important physiological consequences in that the kinase activity triggered by this viral cyclin may abrogate cell cycle checkpoints in addition to those targeted by cellular cyclin D-cdk6 kinase.     

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.     

Degradation of p27(Kip) cdk inhibitor triggered by Kaposi's sarcoma virus cyclin-cdk6 complex.

The Kaposi's sarcoma-associated human herpesvirus 8 (KSHV/HHV8) encodes a protein similar to cellular cyclins. This cyclin is most closely related to cellular D-type cyclins, but biochemically it behaves atypically in various respects. Complexes formed between the viral cyclin and the cyclin-dependent kinase subunit, cdk6, can phosphorylate a wider range of substrates and are resistant to cdk inhibitory proteins. We show here that the KSHV-cyclin-cdk6 complex phosphorylates p27(Kip) on a C-terminal threonine that is implicated in destabilization of this cdk inhibitor. Expression of the viral cyclin in tissue culture cells overcomes a cell cycle block by p27(Kip). However, full cell-cycle transit of these cells appears to depend on C-terminal phosphorylation of p27(Kip) and seems to involve transactivation of other cellular cyclin-dependent kinases. A p27(Kip)-phosphorylating cdk6 complex exists in cell lines derived from primary effusion lymphoma and in Kaposi's sarcoma, this indicating that virally induced p27(Kip) degradation may occur in KSHV-associated tumours.     

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.     

The expression and characterization of cyclin-dependent kinase 6 during the activation of murine G0 T-cells.

Recent evidence strongly suggest that the D type cyclins with cdk4 and cdk6 form holoenzymes that regulate cell cycle events earlier in G1 than cyclin E/cdk2 complexes which functions near the G1/S transition. In human T lymphocytes cdk6 has been shown to be the initial retinoblastoma protein kinase detectable at mid G1. Following activation of splenic derived murine G0T-cells, cdk6, cyclin D2 and D3 specific mRNAs were detected early in G1 and reached maximal levels prior to or near G1/S. The phosphorylation of retinoblastoma protein from T-cells was detected very early in G1 and was associated mainly with cdk6/cyclin D2 complexes which accounted for a minor portion of the total cellular cdk6 contained in the cytoplasmic fraction of T-cells and mostly in the catalytically inactive form.     

Crystal structure of a viral cyclin, a positive regulator of cyclin-dependent kinase 6.

BACKGROUND: Cyclin-dependent kinases (CDKs) have a central role in cell-cycle control and are activated by complex formation with positive regulatory proteins called cyclins and by phosphorylation. The overexpression and mutation of cyclins and CDKs has been associated with tumorigenesis and oncogenesis. A virus-encoded cyclin (v-cyclin) from herpesvirus saimiri has been shown to exhibit highest sequence homology to type D cyclins and specifically activates CDK6 of host cells to a very high degree. RESULTS: We have determined the first X-ray structure of a v-cyclin to 3.0 A resolution. The structure of the core domains is very similar to those of cyclin A and cyclin H from human cells. To understand the structural basis for the v-cyclin specificity for CDK6 and the insensitivity of the complex to inhibitors of the p21 and INK4 families, a v-cyclin-CDK2 model was built on the basis of the known structures of human cyclin A in complex with CDK2 and the CDK inhibitor p27(Kip1). CONCLUSIONS: Although many critical interactions between cyclin A and CDK2 would be conserved in a v-cyclin-CDK2 complex, some appear sterically or electrostatically unfavorable due to shifts in the backbone conformation or sidechain differences and may contribute to v-cyclin selectivity for CDK6. The insensitivity of v-cyclin-CDK6 complexes to inhibitors of the p21 family is probably due to structural changes in v-cyclin that lead to a flatter surface area offering fewer potential contacts with the protein inhibitor. In addition, sequence changes in v-cyclin eliminate hydrogen-bonding partners for atoms of the p27(Kip1) inhibitor. This structure provides the first model for interactions between v-cyclins and host cell-cycle proteins; these interactions may be important for virus survival as well as oncogenic transformation of host cells.     

Identification of G1 kinase activity for cdk6, a novel cyclin D partner.

A family of vertebrate cdc2-related kinases has been identified, and these kinases are candidates for roles in cell cycle regulation. Here, we show that the human PLSTIRE gene product is a novel cyclin-dependent kinase, cdk6. The cdk6 kinase is associated with cyclins D1, D2, and D3 in lysates of human cells and is activated by coexpression with D-type cyclins in Sf9 insect cells. Furthermore, we demonstrate that endogenous cdk6 from human cell extracts is an active kinase which can phosphorylate pRB, the product of the retinoblastoma tumor suppressor gene. The activation of cdk6 kinase occurs during mid-G1 in phytohemagglutinin-stimulated T cells, well prior to the activation of cdk2 kinase. This timing suggests that cdk6, and by analogy its homolog cdk4, links growth factor stimulation with the onset of cell cycle progression.     

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.     

Cyclin D1 is dispensable for G1 control in retinoblastoma gene-deficient cells independently of cdk4 activity.

To elucidate the regulator-versus-target relationship in the cyclin D1/cdk4/retinoblastoma protein (pRB) pathway, we examined fibroblasts from RB-1 gene-deficient and RB-1 wild-type littermate mouse embryos (ME) and in human tumor cell lines that differed in the status of the RB-1 gene. The RB+/+ and RB-/- ME fibroblasts expressed similar protein levels of D-type cyclins, cdk4, and cdk6, showed analogous spectra and abundance of cellular proteins complexed with cdk4 and/or cyclins D1 and D2, and exhibited comparable associated kinase activities. Of the two human cell lines established from the same sarcoma biopsy, the RB-positive SKUT1B cells contained cdk4 that was mainly associated with D-type cyclins, contrary to a predominant cdk4-p16INK4 complex in the RB-deficient SKUT1A cells. Antibody-mediated neutralization of cyclin D1 arrested the RB-positive ME and SKUT1B cells in G1, whereas this cyclin appeared dispensable in the RB-deficient ME and SKUT1A cells. Lack of requirement for cyclin D1 therefore correlated with absence of functional pRB, regardless of whether active cyclin D1/cdk4 holoenzyme was present in the cells under study. Consistent with a potential role of cyclin D/cdk4 in phosphorylation of pRB, monoclonal anti-cyclin D1 antibodies supporting the associated kinase activity failed to significantly affect proliferation of RB-positive cells, whereas the antibody DCS-6, unable to coprecipitate cdk4, efficiently inhibited G1 progression and prevented pRB phosphorylation in vivo. These data provide evidence for an upstream control function of cyclin D1/cdk4, and a downstream role for pRB, in the order of events regulating transition through late G1 phase of the mammalian cell division cycle.     

Viral cyclin-cyclin-dependent kinase 6 complexes initiate nuclear DNA replication

The cyclins encoded by Kaposi sarcoma-associated herpesvirus and herpesvirus saimiri are homologs of human D-type cyclins. However, when complexed to cdk6, they have several activities that distinguish them from D-type cyclin-cdk6 complexes, including resistance to cyclin-dependent kinase inhibitors and an enhanced substrate range. We find that viral cyclins interact with and phosphorylate proteins involved in replication initiation. Using mammalian in vitro replication systems, we show that viral cyclin-cdk6 complexes can directly trigger the initiation of DNA synthesis in isolated late-G(1)-phase nuclei. Viral cyclin-cdk6 complexes share this capacity with cyclin A-cdk2, demonstrating that in addition to functioning as G(1)-phase cyclin-cdk complexes, they function as S-phase cyclin-cdk complexes.     

CAK-independent Activation of CDK6 by a Viral Cyclin

In normal cells, activation of cyclin-dependent kinases (cdks) requires binding to a cyclin and phosphorylation by the cdk-activating kinase (CAK). The Kaposi's sarcoma-associated herpesvirus encodes a protein with similarity to D-type cyclins. This KSHV-cyclin activates CDK6, alters its substrate specificity, and renders CDK6 insensitive to inhibition by the cdk inhibitor p16(INK4a). Here we investigate the regulation of the CDK6/KSHV-cyclin kinase with the use of purified proteins and a cell-based assay. We find that KSHV-cyclin can activate CDK6 independent of phosphorylation by CAK in vitro. In addition, CAK phosphorylation decreased the p16(INK4a) sensitivity of CDK6/KSHV-cyclin complexes. In cells, expression of CDK6 or to a lesser degree of a nonphosphorylatable CDK6(T177A) together with KSHV-cyclin induced apoptosis, indicating that CDK6 activation by KSHV-cyclin can proceed in the absence of phosphorylation by CAK in vivo. Coexpression of p16 partially protected cells from cell death. p16 and KSHV-cyclin can form a ternary complex with CDK6 that can be detected by binding assays as well as by conformational changes in CDK6. The Kaposi's sarcoma-associated herpesvirus has adopted a clever strategy to render cell cycle progression independent of mitogenic signals, cdk inhibition, or phosphorylation by CAK.     

Identification of multiple cell cycle regulatory functions of p57Kip2 in human T lymphocytes

The specific functions of p57(Kip2) in lymphocytes have not yet been fully elucidated. In this study, it is shown that p57(Kip2), which is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors, is present in the nuclei of normal resting (G(0)) T cells from peripheral blood and in the nuclei of the T cell-derived Jurkat cell line. Activation through the TCR results in rapid transport of cytoplasmic cyclin-dependent kinase 6 (cdk6) to nuclei, where it associates with cyclin D and p57(Kip2) in active enzyme complexes. Using purified recombinant proteins, it was shown in vitro that addition of p57(Kip2) protein to a mixture of cyclin D2 and cdk6 enhanced the association of the latter two proteins and resulted in phosphorylation of p57(Kip2). To probe further the function of p57(Kip2), Jurkat cells stably transfected with a plasmid encoding p57(Kip2) under control of an inducible (tetracycline) promoter were made. Induction of p57(Kip2) resulted in increased association of cdk6 with cyclin D3, without receptor-mediated T cell stimulation. The overall amounts of cdk6 and cyclin D3, and also of cdk4 and cyclin E, remained unchanged. Most notably, increased p57(Kip2) levels resulted in marked inhibition of both cyclin E- and cyclin A-associated cdk2 kinase activities and a decrease in cyclin A amounts. Therefore, although facilitating activation of cdk6, the ultimate outcome of p57(Kip2) induction was a decrease in DNA synthesis and cell proliferation. The results indicate that p57(Kip2) is involved in the regulation of several aspects of the T cell cycle.     

Crystal structure of a gamma-herpesvirus cyclin-cdk complex

Several gamma-herpesviruses encode proteins related to the mammalian cyclins, regulatory subunits of cyclin-dependent kinases (cdks) essential for cell cycle progression. We report a 2.5 A crystal structure of a full-length oncogenic viral cyclin from gamma-herpesvirus 68 complexed with cdk2. The viral cyclin binds cdk2 with an orientation different from cyclin A and makes several novel interactions at the interface, yet it activates cdk2 by triggering conformational changes similar to cyclin A. Sequences within the viral cyclin N-terminus lock part of the cdk2 T-loop within the core of the complex. These sequences and others are conserved amongst the viral and cellular D-type cyclins, suggesting that this structure has wider implications for other cyclin-cdk complexes. The observed resistance of this viral cyclin-cdk complex to inhibition by the p27(KIP:) cdk inhibitor is explained by sequence and conformational variation in the cyclin rendering the p27(KIP:)-binding site on the cyclin subunit non-functional.     

Inhibition of p53-mediated growth arrest by overexpression of cyclin-dependent kinases.

Rat fibroblasts transformed by a temperature-sensitive mutant of murine p53 undergo a reversible growth arrest in G1 at 32.5 degrees C, the temperature at which p53 adopts a wild-type conformation. The arrested cells contain inactive cyclin-dependent kinase 2 (cdk2) despite the presence of high levels of cyclin E and cdk-activating kinase activity. This is due in part to p53-dependent expression of the p2l cdk inhibitor. Upon shift to 39 degrees C, wild-type p53 is lost and cdk2 activation and pRb phosphorylation occur concomitantly with loss of p2l. This p53-mediated growth arrest can be abrogated by overexpression of cdk4 and cdk6 but not cdk2 or cyclins, leading to continuous proliferation of transfected cells in the presence of wild-type p53 and p2l. Kinase-inactive counterparts of cdk4 and cdk6 also rescue these cells from growth arrest, implicating a noncatalytic role for cdk4 and cdk6 in this resistance to p53-mediated growth arrest. Aberrant expression of these cell cycle kinases may thus result in an oncogenic interference with inhibitors of cell cycle progression.     

Identification and properties of an atypical catalytic subunit (p34PSK-J3/cdk4) for mammalian D type G1 cyclins.

Murine D type cyclins associate with a catalytic subunit (p34PSK-J3) with properties distinct from known cyclin-dependent kinases (cdks). Mouse p34PSK-J3 shows less than 50% amino acid identity to p34cdc2, p33cdk2, and p36cdk3, lacks a PSTAIRE motif, and does not bind to p13suc1. Cyclin D1-p34PSK-J3 complexes accumulate in macrophages during G1 and decline in S phase, whereas complexes involving cyclins D2 and D3 form in proliferating T cells. Although histone H1 kinase activity is not detected in cyclin D or PSK-J3 immunoprecipitates, cyclin D-p34PSK-J3 complexes assembled in vitro stably bind and phosphorylate the retinoblastoma gene product (pRb) and an Rb-like protein (p107) but do not interact with pRb mutants that are functionally inactive. Thus, p34PSK-J3 is a cyclin D-regulated catalytic subunit that acts as an Rb (but not H1) kinase.     

Evidence for coordinated interaction of cyclin D3 with p21 and cdk6 in directing the development of uterine stromal cell decidualization and polyploidy during implantation.

Uterine decidualization, characterized by stromal cell proliferation, and differentiation into specialized type of cells (decidual cells) with polyploidy, during implantation is critical to the pregnancy establishment in mice. The mechanisms by which the cell cycle events govern these processes are poorly understood. The cell cycle is tightly regulated at two particular checkpoints, G1-S and G2-M phases. Normal operation of these phases involves a complex interplay of cyclins, cyclin-dependent kinases (cdks) and cdk inhibitors (CKIs). We previously observed that upregulation of uterine cyclin D3 at the implantation site is tightly associated with decidualization in mice. To better understand the role of cyclin D3 in this process, we examined cell-specific expression and associated interactions of several cell cycle regulators (cyclins, cdks and CKIs) specific to different phases of the cell cycle during decidualization in mice. Among the various cell cycle molecules examined, coordinate expression and functional association of cyclin D3 with cdk4 suggest a role for proliferation and, that of cyclin D3 with p21 and cdk6 is consistent with the development of polyploidy during stromal cell decidualization.     

Cyclin-cyclin-dependent kinase regulatory response is linked to substrate recognition

Cyclin/cyclin-dependent kinase (CDK) complexes are critical regulators of cellular proliferation. A complex network of regulatory mechanisms has evolved to control their activity, including activating and inactivating phosphorylation of the catalytic CDK subunit and inhibition through specific regulatory proteins. Primate herpesviruses, including the oncogenic Kaposi sarcoma herpesvirus, encode cyclin D homologues. Viral cyclins have diverged from their cellular progenitor in that they elicit holoenzyme activity independent of activating phosphorylation by the CDK-activating kinase and resistant to inhibition by CDK inhibitors. Using sequence comparison and site-directed mutagenesis, we performed molecular analysis of the cellular cyclin D and the Kaposi sarcoma herpesvirus-cyclin to delineate the molecular mechanisms behind their different behavior. This provides evidence that a surface recognized for its involvement in the docking of CIP/KIP inhibitors is required and sufficient to modulate cyclin-CDK response to a range of regulatory cues, including INK4 sensitivity and CDK-activating kinase dependence. Importantly, amino acids in this region are critically linked to substrate selection, suggesting that a mutational drift in this surface simultaneously affects function and regulation. Together our work provides novel insight into the molecular mechanisms governing cyclin-CDK function and regulation and defines the biological forces that may have driven evolution of viral cyclins.