MK2 and MK3 represent protein kinases downstream of p38 mitogen-activated protein kinase (MAPK).Deletion of the MK2 gene in mice resulted in an impaired inflammatory response although MK3, which displays extensive structural similarities and identical functional properties in vitro, is still present. Here, we analyze tumor necrosis factor (TNF) production and expression of p38 MAPK and tristetraprolin (TTP) in MK3-deficient mice and demonstrate that there are no significant differences with wild-type animals. We show that in vivo MK2 and MK3 are expressed and activated in parallel. However, the level of activity of MK2 is always significantly higher than that of MK3. Accordingly, we hypothesized that MK3 could have significant effects only in an MK2-free background and generated MK2/MK3 double-knockout mice. Unexpectedly, these mice are viable and show no obvious defects due to loss of compensation between MK2 and MK3. However, there is a further reduction of TNF production and expression of p38 and TTP in double-knockout mice compared to MK2-deficient mice. This finding, together with the observation that ectopically expressed MK3 can rescue MK2 deficiency similarly to MK2, indicates that both kinases share the same physiological function in vivo but are expressed to different levels.Downstream of mitogen-activated protein kinases (MAPKs) different groups of MAPK-activated protein kinases (MAP KAPKs) have been defined (reviewed in reference 28). These enzymes transduce signals to target proteins that are not direct substrates of the MAPKs and, therefore, serve to relay phosphorylation-dependent signaling within MAPK cascades to diverse cellular functions. One of these groups is formed by the three MAPKAPKs, MK2, MK3 (also known as 3pK), and MK5 (also designated PRAK) (reviewed in reference 12). While MK5 is mainly activated by the atypical MAPK ERK3 (29, 30), the remaining two kinases, MK2 and MK3, are directly downstream of the MAPK p38␣/␤ (7,10,24,27,31). Phosphorylation of MK2 and MK3 by p38␣/␤ at two or three major regulatory sites leads to activation and coupled nuclear export of both enzymes, which are localized in the nucleus of resting cells (4,8,26,36,41).A wide variety of substrates has been described for MK2 including proteins interacting with the cytoskeleton, such as small heat shock protein Hsp25 (33); mRNA-binding proteins, such as tristetraprolin (TTP) (6, 32); transcription factors, such as heat shock factor 1 (38); and regulators of the cell cycle and apoptosis, such as Cdc25B/C (23). The phosphorylation site recognition motifs of MK2 and MK3 are similar (20) or even identical (7). Despite the similar recognition motif, not all MK2 substrates have been described as MK3 substrates so far, probably because in most cells MK2 activity dominates and makes analysis of the minor MK3 activity dependent on antibodies which discriminate between both enzymes (7).MK2-deficient mice are more resistant than wild type to endotoxic shock due to impaired production of cytokines such as tumor necrosis factor (T...

SHPTP2 is a ubiquitously expressed tyrosine-specific protein phosphatase that contains two aminoterminal Src homology 2 (SH2) domains responsible for its association with tyrosine-phosphorylated proteins. In this study, expression ofdominant interfering mutants of SHPTP2 was found to inhibit insulin stimulation of c-fos reporter gene expression and activation of the 42-kDa (Erk2) and 44-kDa (Erkl) mitogen-activated protein kinases. Cotransfection of dominant interfering SHPTP2 mutants with v-Ras or Grb2 indicated that SHPTP2 regulated insulin signaling either upstream of or in parallel to Ras function. Furthermore, phosphotyrosine blotting and immunoprecipitation identified the 125-kDa focal adhesion kinase (pp125FAK) as a substrate for insulin-dependent tyrosine dephosphorylation. These data demonstrate that SHPTP2 functions as a positive regulator of insulin action and that insulin signaling results in the dephosphorylation of tyrosine-phosphorylated ppl25FAK.The insulin receptor is a ligand-stimulated transmembrane protein-tyrosine kinase that phosphorylates itself as well as intracellular substrates on specific tyrosine residues (1, 2). One proximal intracellular target for the kinase-activated insulin receptor has been identified as a 185-kDa protein, termed insulin receptor substrate 1 (IRS1) (3,4). This molecule contains several insulin receptor-specific tyrosine phosphorylation sites that provide recognition signals for the binding of specific Src homology 2 (SH2) domain-containing proteins (5, 6). The interaction of IRS1 with signaling proteins containing multiple SH2 domains provides a mechanism by which insulin can modulate the function of several distinct pathways. For example, activation of phosphatidylinositol (PI) 3-kinase activity occurs upon the association of the p85 subunit of the PI 3-kinase with tyrosine-phosphorylated IRS1 (7-9). It has been suggested that the insulin activation of Ras function results from the interaction and/or appropriate targeting of the guanine nucleotide exchange factor Sos with IRS1 (6, 10). This is thought to occur via the constitutive association of the Src homology 3 (SH3) domains of the adapter protein Grb2 with the C-terminal domain of Sos (Grb2/Sos complex) and subsequent binding of the Grb2 SH2 domain to tyrosinephosphorylated IRS1 (10, 11).Recently, several groups have identified a ubiquitously expressed 68-kDa tyrosine-specific protein phosphatase, SHPTP2 (also termed Syp, PTP1D, SHPTP3, PTP2C, or PTPL1), that contains two N-terminal SH2 domains and a C-terminal catalytic domain (12-17). The SH2 domains of this phosphatase mediate the binding of SHPTP2 to tyrosinephosphorylated epidermal growth factor receptor, plateletderived growth factor receptor, and IRS1, resulting in the activation of protein-tyrosine-phosphatase activity (18)(19)(20)(21). Although protein tyrosine phosphatase activity is generally thought to function as the inactivating arm of receptor kinase signaling pathways, in the case of T-cell receptor signaling, the CD45 protein-tyrosi...

Expression of the major heat shock and stress-induced protein, HSP70, is under complex regulatory control in human cells. In addition to being induced by physiological stress such as heat shock or transition metals, the HSP70 gene is induced by serum stimulation and immortalizing products of the adenovirus ElA 13S and polyoma large tumor antigen genes. Here we show that expression of the human HSP70 gene is tightly regulated during the cell cycle. Using selective mitotic detachment, a noninductive method to obtain synchronous populations of HeLa cells, we show that levels of HSP7O mRNA rapidly increase 10-to 15-fold upon entry into S phase and decline by late S and G2. A transient increase in HSP70 synthesis is detected during early S phase. The subcellular localization of HSP70 varies throughout the cell cycle; the protein is diffusely distributed in the nucleus and cytoplasm in G1, localized in the nucleus in S, and again diffusely distributed in G2 cells. We suggest that the temporal pattern of HSP7O expression during S phase, the nuclear localization, and activation by trans-acting immortalizing proteins indicate a role for HSP7O in the nucleus of replicating cells.The cellular transition from the resting state to the growing state requires expression of certain genes whose products regulate critical events during the Go and G, phases of the cell cycle. Serum stimulation has been widely used as a method to identify and examine genes that are growth-regulated. The addition of fresh serum to quiescent mammalian cells activates the expression of a family of growth-regulated genes, including c-fos, c-myc, P-interferon, proliferin, p53, and HSP70 (1-10). Many of these genes are activated by specific growth factors such as platelet-derived growth factor or epidermal growth factor, suggesting the possibility that their expression may be regulated by common mechanisms (1, 2, 7).Workers in our laboratory have studied the expression of the major heat shock protein, HSP70, in human cells. An intriguing relationship between the expression of heat shock proteins and cellular transformation has been established. HSP70 is expressed at high levels in transformed mammalian cells and is induced in cells infected with DNA tumor viruses (11-15). Expression from the HSP70 promoter is induced by the ElA 13S product of adenovirus and by large tumor antigen of polyoma (15,16 We have demonstrated (10) that HSP70 gene expression in a transformed human cell line, HeLa, was induced by serum stimulation. To examine whether this corresponds to expression at a specific point during the cell cycle or to activation by specific growth factors, we have used a noninductive method-that of selective mitotic detachment-to obtain synchronous populations of cells. We show that maximal levels of HSP70 mRNA are detected in S phase at the peak of DNA synthesis. HSP70 protein synthesis increases transiently at this point and increased levels of HSP70 are detected in the nucleus of S-phase cells. METHODSCell Culture and Synchronization. HeLa cel...

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Introduction The receptor for advanced glycation end products (RAGE), a multi-ligand member of the immunoglobulin superfamily, contributes to acute and chronic disease processes, including sepsis.