The large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is a multifunctional protein that contains a serine-threonine protein kinase (PK) activity (Nelson, J. W., Zhu, J., Smith, C. C., Kulka, M., and Aurelian, L. (1996) J. Biol. Chem. 271, 17021-17027). Phylogenetic analyses indicated that ICP10 PK belongs to a distinct subfamily of growth factor receptor serine-threonine PKs that are characterized by their ability to function with a limited number of conserved catalytic motifs (Hunter, J. C. R., Smith, C. C., and Aurelian, L. (1995) Int. J. Onc. 7, 515-522). Here, we report the isolation and characterization of a novel gene, designated H11, that contains an open reading frame of 588 nucleotides, which encodes a protein similar to ICP10 PK. The H11 protein has Mn 2؉ -dependent serinethreonine-specific PK activity as determined with a GST-H11 fusion protein and by immununocomplex PK/ immunoblotting assays of 293 cells transfected with a H11 eukaryotic expression vector. PK activity is ablated by mutation of Lys 113 within the presumtive catalytic motif II (invariant Lys). 293 cells stably transfected with H11 acquire anchorage-independent growth. Endogenous H11 RNA and the H11 phosphoprotein are expressed in melanoma cell lines and primary melanoma tissues at levels higher than in normal melanocytes and in benign nevi. Melanoma cell proliferation is inhibited by treatment with antisense oligonucleotides that inhibit H11 translation, suggesting that H11 expression is associated with cell growth.Several herpes viruses including herpes simplex virus type 1 (HSV-1) 1 and herpes simplex virus type 2 (HSV-2) express a distinct ribonucleotide reductase activity formed by the association of a large (R1) and a small (R2) subunit. The HSV-2 R1 gene (also known as ICP10) differs from its counterparts in eukaryotic and prokaryotic cells and in other viruses in that it possesses a unique one-third 5Ј-terminal domain (1) that codes for a serine (Ser)-threonine (Thr) protein kinase (PK) (2-8) and causes neoplastic transformation of immortalized cells (9 -11). Unlike other known PKs that have at least 12 conserved catalytic motifs (12, 13), ICP10 PK functions with only eight such motifs. They are clustered close to the N terminus, downstream of a transmembrane (TM) domain and surround a Src homology region 3-binding module (2,4,6,14). The ICP10 PK activity is Mn 2ϩ ion-dependent, does not require monovalent cations, and is not inhibited by zinc sulfate, properties distinct from those of many cellular kinases such as casein kinase II (5). ICP10 is located on the cell surface and its PK activity is intrinsic. Mutants in which the TM domain or the conserved PK catalytic motifs were deleted or otherwise altered were rendered PK negative (4,5,7,8), and the PK activity of a chimeric protein consisting of ICP10 PK and the ligand-binding domain of the epidermal growth factor receptor was activated by epidermal growth factor (15). Most significantly, the ICP10 PK activity was ablated by mutation of the invarian...

Signaling pathways, the ultimate targets of which are nuclear transcription factors, determine the cell's ability to respond to external stimuli. Transduced signals can be interpreted as mitogenic-proliferative, differentiating, or apoptotic, depending on the cell type and the nature and duration of the stimulus. The mitogenic Ras/MEK/MAPK pathway is initiated by growth factor mediated activation of cognate receptors on the cell surface causing the membrane bound G protein Ras to adopt an active, GTP-bound state. Ras coordinates the activation of a cascade of serine (Ser)-threonine (Thr) kinases that begins with Raf and is followed by MAP kinase kinase 1 and 2 (MEK1/2) and mitogen-activated protein kinase (MAPK1/2) and culminates in the expression of the transcription factor c-Fos (41, 46, 48, 67) which is important for promoting cell cycle progression into S phase (14, 39, 44). The GTPase-activating protein Ras-GAP, a major negative regulator of Ras activity, acts to enhance the weak intrinsic Ras GTPase activity by accelerating the hydrolysis rate of bound GTP to GDP (7,27,31,48,57). Ras-GAP inactivation, for example by phosphorylation on Ser-Thr residues, has been implicated in Ras activation (11,24,66,89). The specificity of the signal transduction is determined by protein domains such as SH2, SH3, and PH that bind unique motifs in target proteins for recruitment into signaling complexes (55, 63).Viruses depend on cells for their replication. They take advantage of preexisting signaling pathways or activate them through various strategies, including activation of the RAS/ MEK/MAPK pathway by Ras-GAP inactivation (32). Vaccinia virus (42), simian virus 40 (SV40) (77), human immunodeficiency virus (HIV) type 1 (35), herpesvirus saimiri (38), and coxsackievirus B3 (32) depend upon the activated RAS/MEK/ MAPK pathway for growth. Herpes simplex virus type 1 (HSV-1) increases the levels of transcription factors c-Jun (37) and 23), and its replication is enhanced by activation of the c-Jun N-terminal kinase (JNK) and p38 of the stress-activated signal pathway (58,88). By contrast, HSV-2 increases c-Fos transcription (26), suggesting that the two HSV serotypes use different strategies to take advantage of signaling pathways. However, the mechanism responsible for c-Fos induction in HSV-2-infected cells, the contribution of the Ras/ MEK/MAPK pathway, and their relationship to virus replication are still unknown.The large subunits of HSV-1 and HSV-2 ribonucleotide reductase (RR1) differ from their counterparts in eukaryotic and prokaryotic cells and in other viruses in that they have an intrinsic PK activity (2,5,12,15,17,49,50,60,65). The RR1 promoter has an octamer-TAATGARAT sequence that responds to the VP16-Oct1 complex (18,78,86,87). RR1 is expressed with apparently biphasic kinetics that consist of immediate-early (IE; also known as ␣) and early components (3,30,84,86,87,90). Expression is independent of the regulatory IE protein ICP4 (18,78,86,87). AP-1 cognate sites in the HSV-2 RR1 (also known as ICP10) promo...

H11, the eukaryotic homologue of a herpes simplex virus protein, has the crystallin motif of heat shock proteins (Hsp), but it differs from canonical family members in that mRNA and protein levels were reduced in various tumor tissues and cell lines (viz. melanoma, prostate cancer and sarcoma) relative to their normal counterparts. In these cells, expression was not restored by heat shock, but rather by the demethylating agent 5-aza-2-deoxycytidine (Aza-C). Forced H11 expression by Aza-C treatment, transient transfection with H11 expression vectors, or retrovirus-mediated delivery of H11 under the control of a tetracycline-sensitive promoter triggered apoptosis. This is evidenced by a significant (p < 0.001) increase in the percentage of cells positive for terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and for activation of caspase-3 and p38MAPK and by the co-localization of TUNEL ؉ nuclei with increased H11 levels. Apoptosis was partially inhibited by the pancaspase inhibitor benzyloxycarbonylVal-Ala-Asp-fluoromethyl ketone or the p38MAPK inhibitor SB203580. It was abrogated by co-treatment with both inhibitors, suggesting that H11-triggered apoptosis is both caspase-and p38MAPK-dependent. A single site mutant (H11-W51C) had cytoprotective activity related to MEK/ERK activation, and it blocked H11-induced apoptosis in co-transfected and Aza-C-treated cells, indicating that it is a dominant negative mutant. This is the first report of a heat shock protein with proapoptotic activity.

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