Activation of growth factor receptor protein tyrosine kinases frequently results in the binding of numerous proteins to their tyrosine-phosphorylated cytoplasmic domains. These interactions involve the SH2 domains of the binding proteins and phosphorylated tyrosines on the receptor molecules, with the specificity of interaction dictated by the amino acid composition surrounding the phosphorylated tyrosine. In the case of the platelet-derived growth factor (PDGF) receptor, the major binding site for Src family tyrosine kinases is in the juxtamembrane domain and includes tyrosine 579 (Mori, S., Rönnstrand, L., Yokote, K., Engström, A., Courtneidge, S. A., Claesson-Welsh, L., and Heldin, C-H. (1993) EMBO J. 12, 2257-2264). To analyze in more detail which amino acids surrounding the phosphorylated tyrosine at position 579 were important for high affinity interaction with Src family kinases, we synthesized a series of phosphopeptides corresponding to this binding site in which single amino acids were individually changed and tested their ability to compete with the PDGF receptor for binding of Fyn. We found that not only the three residues carboxyl-terminal to the phosphorylated tyrosine were important but that also residues at positions -1 and +4 relative to the tyrosine were required. Phosphorylation of both tyrosines 579 and 581 significantly increased competition efficiency. The activated colony stimulating factor-1 (CSF-1) receptor, which is known to associate with Src family kinases, has a sequence in its juxtamembrane region similar to that surrounding Tyr-579 of the PDGF receptor, and a phosphopeptide modeled on this sequence competed the association of Fyn with the receptor in vitro. Furthermore, mutational analysis demonstrated that these sequences were required for the efficient association of Src family kinases with the activated CSF-1 receptor in vivo. Phosphopeptides corresponding to the Src family binding sites of both PDGF and CSF-1 receptors activated Src kinase activity in vitro. These observations support a model in which the enzymatic activity of Src family tyrosine kinases is controlled by intra- and intermolecular interactions of tyrosine-phosphorylated peptides with the SH2 domain of the kinases.

p38alpha MAP kinase is activated in response to many cellular stresses and also regulates the differentiation and/or survival of various cell types in vitro, including skeletal muscle cells and cardiomyocytes. Here we show that targeted inactivation of the mouse p38alpha gene results in embryonic lethality at midgestation correlating with a massive reduction of the myocardium and malformation of blood vessels in the head region. However, this defect appears to be secondary to insufficient oxygen and nutrient transfer across the placenta. When the placental defect was rescued, p38alpha(-/-) embryos developed to term and were normal in appearance. Our results indicate that p38alpha is required for placental organogenesis but is not essential for other aspects of mammalian embryonic development.

Two chromatographically distinct stress‐activated protein kinase kinases (SAPKKs) have been identified in several mammalian cells, termed SAPKK2 and SAPKK3, which activate the MAP kinase family member RK/p38 but not JNK/SAPK in vitro. Here we demonstrate that SAPKK2 is identical or very closely related to the MAP kinase kinase family member MKK3. However, under our assay conditions, SAPKK3 was the major activator of RK/p38 detected in extracts prepared from stress‐ or interleukin‐1‐stimulated epithelial (KB) cells, from bacterial lipopolysaccharide and tumour necrosis factor alpha‐stimulated THP1 monocytes or from rabbit skeletal muscle. The activated form of SAPKK3 was purified from muscle to near homogeneity, and tryptic peptide sequences were used to clone human and murine cDNAs encoding this enzyme. Human SAPKK3 comprised 334 amino acids and was 78% identical to MKK3. The murine and human SAPKK3 were 97% identical in their amino acid sequences. We also cloned a different murine cDNA that appears to encode a SAPKK3 protein truncated at the N‐terminus. SAPKK3 is identical to the recently cloned MKK6.

The Src-like adaptor protein (Slap) is a recently identified adaptor protein containing Src homology 3 (SH3) and SH2 domains. Slap is found in a wide range of cell types and was shown to interact with the Eck receptor tyrosine kinase in a yeast two-hybrid interaction screen [1]. Here, we found that Slap is expressed in NIH3T3 cells and could associate with the activated platelet-derived growth factor (PDGF) receptor. Using mutated versions of the PDGF receptor and phosphopeptide competition experiments, we determined that Slap has the highest affinity for the Src-binding site of the PDGF receptor. Our inability to produce cell lines that stably expressed Slap suggested that Slap inhibited cell growth. We further investigated this issue by transiently expressing Slap by microinjection. Overexpression of Slap by this method inhibited DNA synthesis induced by PDGF and serum, whereas overexpression of the adaptor proteins Grb2 and Shc did not. Finally, microinjection of a Slap antibody into NIH3T3 cells that had been stimulated with suboptimal doses of growth factors potentiated the effects of the growth factors. These data suggest that, unlike other adaptor proteins, Slap is a negative regulator of signalling initiated by growth factors.

We have investigated the ability of the mitogen-activated protein kinase (MAPK) kinase MKK6 to activate different members of the p38 subfamily of MAPKs and found that some MKK6 mutants can efficiently activate p38␣ but not p38␥. In contrast, a constitutively active MKK6 mutant activated both p38 MAPK isoforms to similar extents. The same results were obtained upon coexpression in Xenopus oocytes and in vitro using either MKK6 immunoprecipitates from transfected cells or bacterially produced recombinant proteins. We also found that the preferential activation of p38␣ by MKK6 correlated with more efficient binding of MKK6 to p38␣ than to p38␥. Furthermore, increasing concentrations of constitutively active MKK6 differentially activated either p38␣ alone (low MKK6 activity) or both p38␣ and p38␥ (high MKK6 activity), both in vitro and in injected oocytes. The determinants for selectivity are located at the carboxyl-terminal lobe of p38 MAPKs but do not correspond to the activation loop or common docking sequences. We also showed that different stimuli can induce different levels of endogenous MKK6 activity that correlate with differential activation of p38 MAPKs. Our results suggest that the level of MKK6 activity triggered by a given stimulus may determine the pattern of downstream p38 MAPK activation in the particular response.Cellular responses to many external stimuli involve the activation of several types of mitogen-activated protein kinase (MAPK) 1 signaling pathways. Three major subfamilies of MAPKs have been described in vertebrates. The p42/p44 ERK MAPKs are mainly activated by growth factors and other stimuli involved in cell proliferation and differentiation processes. In contrast, the SAPK/JNK and the p38 MAPKs are strongly activated in response to stress conditions and proinflammatory cytokines. Despite the diversity in function and upstream signaling events, MAPKs are always activated by a highly conserved mechanism that involves phosphorylation on both a Thr and a Tyr residue catalyzed by a MAPK kinase. The phosphorylation motif Thr-Xaa-Tyr is located in the so called activation loop or T loop whose amino acid sequence varies among different MAPK subfamilies. Accordingly, there are different activating MAPK kinases that in most cases are specific for each subgroup of MAPKs (reviewed by Refs. 1-3) The p38 MAPK subfamily plays important roles in cytokine production and the stress response (reviewed by Ref. 4). Recent reports have also demonstrated additional functions for p38 MAPKs, for example, in the inhibition of cell cycle progression, in developmental processes such as egg polarity and wing morphogenesis in Drosophila, and in the differentiation of several vertebrate cell types including neurons, adipocytes and myoblasts (reviewed in Ref. 5). Four p38 MAPKs have been cloned so far in higher eukaryotes: p38␣/XMpk2/CSBP (6 -9), p38␤/ p38␤2 (10, 11), p38␥/SAPK3/ERK6 (12-14), and p38␦/SAPK4 (15-17). These four proteins are 60 -70% identical in their amino acid sequence and are all activated by the MA...

The Src family of protein tyrosine kinases has been implicated in the response of cells to platelet-derived growth factor (PDGF) or epidermal growth factor (EGF). We recently described a microinjection approach that we used to demonstrate that kinase activity of Src family members is required for PDGF-and EGF-induced S-phase entry of fibroblasts. We have now used this approach to ask whether a functional SH3 domain of Src is required to transduce the mitogenic signal upon PDGF or EGF stimulation. Microinjection of plasmids encoding Src mutants lacking the SH3 domain (Src⌬SH3) or point-mutated within the ligand binding surface of the SH3 domain, but with intact kinase domains, inhibited the mitogenic effect of PDGF and EGF in fibroblasts. Src⌬SH3 could still associate with the PDGF receptor, suggesting that the inhibitory effect of the Src SH3 mutants was brought about by a failure of the PDGF receptor⅐Src⌬SH3 complex to relay the mitogenic signal further downstream. Chimeric molecules in which the Src SH3 domain was replaced with that of spectrin or Lck also blocked PDGF-induced DNA synthesis, whereas a chimera containing the Fyn SH3 domain did not. These data suggest that the Src or Fyn SH3 domain is required either for correct substrate selection or to recruit other proteins to the PDGF receptor.

The human tumor marker protein p150 was identified as the largest subunit of eukaryotic translation initiation factor 3 (eIF3) (also known as p170/p180). Its expression level is not only upregulated in many transformed cell lines, but also in several human cancers including breast, cervical, esophageal, and stomach carcinomas. The function of p150 in cancer and initiation of translation are not well understood. Using the yeast two-hybrid genetic screen, we found that a portion of p150 interacts with hPrt1, another subunit of eIF3, and cytokeratin 7, an intermediate filament protein. The interactions between p150 and hPrt1, and between p150 and cytokeratin 7 were verified both in vivo and in vitro. The interaction site for hPrt1 was mapped to the carboxyl half of the coiled-coil region of the p150 protein between amino acids 664-835. The expression of hPrt1 was clearly upregulated in cancer tissue, similarly to that of p150. By contrast, no substantial difference in the expression level of cytokeratin 7 was observed between cancer and normal breast tissue, suggesting that cytokeratin 7 expression is not co-regulated with p150. Taken together, our studies suggest a new role for p150 in translation initiation, possibly by acting as an adapter molecule between the translation initiation apparatus and the cytoskeleton structure in the cell.

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