No abstract

We have identified two components of a new protein kinase signaling cascade, MAPK/ERK kinase 5 (MEK5) and extracellular signal-regulated kinase 5 (ERK5). The MEK5 cDNA was isolated by degenerate PCR and encodes a 444-amino acid protein, which has approximately 40% identity to known MEKs. ERK5 was identified by a specific interaction with the MEK5 mutants S311A/T315A and K195M in the yeast two-hybrid system. The proteins were found to interact in an in vitro binding assay as well. ERK5 did not interact with MEK1 or MEK2. ERK5 is predicted to contain 815 amino acids and is approximately twice the size of all known ERKs. The C terminus of ERK5 has sequences which suggest that it may be targeted to the cytoskeleton. Sequences located in the N terminus of MEK5 may be important in coupling GTPase signaling molecules to the MEK5 protein kinase cascade. Both MEK5 and ERK5 are expressed in many adult tissue and are abundant in heart and skeletal muscle. A recombinant GST-ERK5 kinase domain displays autophosphorylation on Ser/Thr and Tyr residues.

Serotonin has been implicated in the regulation of a wide range of brain functions involving alternative behavioral states, including the control of mood, aggression, sex, and sleep. Here, we report that in the nematode Caenorhabditis elegans, serotonin controls a switch between two distinct, on/off states of egg-laying behavior. Through quantitative analysis of the temporal pattern of egg-laying events, we determined that egg laying can be modeled as a novel random process, in which animals fluctuate between discrete behavioral states: an active state, during which eggs are laid in clusters, and an inactive state, during which eggs are retained. Single-cell ablation experiments indicate that two pairs of motor neurons, HSNL/HSNR and VC4/VC5, can induce the active phase by releasing serotonin. These neurons also release acetylcholine, which appears to trigger individual egg-laying events within the active phase. Genetic experiments suggest that determination of the behavioral states observed for C. elegans egg laying may be mediated through protein kinase C-dependent (PKC-dependent) modulation of voltage-gated calcium channels.

No abstract

SHP-1 is a cytosolic protein-tyrosine phosphatase that behaves as a negative regulator in eukaryotic cellular signaling pathways. To understand its regulatory mechanism, we have determined the crystal structure of the C-terminal truncated human SHP-1 in the inactive conformation at 2.8-Å resolution and refined the structure to a crystallographic R-factor of 24.0%. The three-dimensional structure shows that the ligand-free SHP-1 has an auto-inhibited conformation. Its N-SH2 domain blocks the catalytic domain and keeps the enzyme in the inactive conformation, which supports that the phosphatase activity of SHP-1 is primarily regulated by the N-SH2 domain. In addition, the C-SH2 domain of SHP-1 has a different orientation from and is more flexible than that of SHP-2, which enables us to propose an enzymatic activation mechanism in which the C-SH2 domains of SHPs could be involved in searching for phosphotyrosine activators.Tyrosine phosphorylation is a key mechanism for regulating eukaryotic cellular signaling pathways. The protein tyrosine phosphorylation level is precisely regulated by two types of enzymes: protein-tyrosine kinases (PTKs) 1 and protein-tyrosine phosphatases (PTPs), in which PTPs act to counter-balance the process through dephosphorylation of the phosphorylated tyrosines (1, 2). PTPs can be divided into two groups, receptor protein-tyrosine phosphatases and cytosolic proteintyrosine phosphatases. The SH2 domain-containing PTPs, SHP-1 and SHP-2, are both cytosolic PTPs and share many structural and regulatory features. They both have two tandem SH2 domains at the N terminus followed by a single catalytic domain and an inhibitory C-terminal tail. However, irrespective of similar structural and regulatory characteristics, these two enzymes have different biological function in vivo.Different from SHP-2, which is expressed in all kinds of tissues, SHP-1 is predominantly expressed in hematopoietic and epithelial cells and behaves mainly as a negative regulator of signaling pathways in lymphocytes (1, 2). SHP-1 is dormant in the cytosol, with its phosphatase activity inhibited by both the SH2 domains and the C-terminal tail (1,(3)(4)(5). In response to an activation signal, SHP-1 is recruited to membrane-bound inhibitory receptors via the binding of its SH2 domains to the tyrosine-phosphorylated immunoreceptor tyrosine-based inhibitory motif within the cytoplasmic domain of a receptor (6 -8). During this process, SHP-1 undergoes a structural rearrangement, exposes its active site, and binds to the downstream substrates, thereby dephosphorylating the substrates to turn off the cellular signals.SHP-1 also presents in several types of non-hematopoietic cells (9 -12). Overexpression of a catalytically inactive SHP-1 mutant in these cells strongly suppressed mitogen-activated pathways, reducing signal transduction and activation of transcription; these findings demonstrate that SHP-1 has a positive effect on mitogenic signaling in these non-hematopoietic cells (10, 11). Thus, SHP-1 probably has both the nega...