Teri G. Boulton

ERKs: A family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF

We recently described the purification and cloning of extracellular signal-regulated kinase 1 (ERK1), which appears to play a pivotal role in converting tyrosine phosphorylation into the serine/threonine phosphorylations that regulate downstream events. We now describe cloning and characterization of two ERK1-related kinases, ERK2 and ERK3, and provide evidence suggesting that there are additional ERK family members. At least two of the ERKs are activated in response to growth factors; their activations correlate with tyrosine phophorylation, but also depend on additional modifications. Transcripts corresponding to the three cloned ERKs are distinctly regulated both in vivo and in a differentiating cell line. Thus, this family of kinases may serve as intermediates that depend on tyrosine phosphorylation to activate serine/threonine phosphorylation cascades. Individual family members may mediate responses in different developmental stages, in different cell types, or following exposure to different extracellular signals.

Choice of STATs and other substrates specified by modular tyrosine-based motifs in cytokine receptors.

Many members of the cytokine receptor superfamily initiate intracellular signaling by activating members of the Jak family of tyrosine kinases. Activation of the same Jaks by multiple cytokines raises the question of how these cytokines activate distinct intracellular signaling pathways. Selection of particular substrates--the transcriptional activator Stat3 and protein tyrosine phosphatase PTP1D--that characterize responses to the ciliary neurotrophic factor-interleukin-6 cytokine family depended not on which Jak was activated, but was instead determined by specific tyrosine-based motifs in the receptor components--gp130 and LIFR--shared by these cytokines. Further, these tyrosine-based motifs were modular, because addition of a Stat3-specifying motif to another cytokine receptor, that for erythropoietin, caused it to activate Stat3 in a ligand-dependent fashion.

CNTF and LIF act on neuronal cells via shared signaling pathways that involve the IL-6 signal transducing receptor component gp130

Ciliary neurotrophic factor (CNTF) has a variety of actions within the nervous system. While some of the actions of leukemia inhibitory factor (LIF) on neurons resemble those of CNTF, LIF also has broad actions outside of the nervous system that in many cases mimic those of interleukin-6 (IL-6). Comparison of the tyrosine phosphorylations and gene activations induced by CNTF and LIF in neuron cell lines reveals that they are indistinguishable and also very similar to signaling events that characterize LIF and IL-6 responses in hematopoietic cells. We provide a basis for the overlapping actions of these three factors by demonstrating that the shared CNTF and LIF signaling pathways involve the IL-6 signal transducing receptor component gp130. Thus, the receptor system for CNTF is surprisingly unlike those used by the nerve growth factor family of neurotrophic factors, but is instead related to those used by a subclass of hematopoietic cytokines.

Trophic effect of ciliary neurotrophic factor on denervated skeletal muscle

The actions and receptor for ciliary neurotrophic factor (CNTF) are largely restricted to cells of the nervous system, although one of the CNTF receptor components, CNTFR alpha, is expressed by skeletal muscle. Here we show that the other CNTF receptor components, LIFR beta and gp130, are also expressed by skeletal muscle and that expression of all three CNTF receptor components is greatly increased in denervated muscle. In vivo, administration of CNTF activates these receptors on skeletal muscle by inducing receptor phosphorylation and immediate-early gene responses. Furthermore, CNTF reduces the denervation-induced atrophy of muscle and attenuates the reduced twitch and tetanic tensions that result from muscle denervation. Our findings reveal that, in addition to its known neurotrophic actions, CNTF exerts myotrophic effects by attenuating the morphological and functional changes associated with denervation of rat skeletal muscle.

Activation of p42 MAP kinase and the release of oocytes from cell cycle arrest.

Clam oocytes are arrested naturally at the G2/M border in meiosis and contain an inactive 42 kDa ERK/MAP kinase, p42MAPK. Following fertilization, p42MAPK is rapidly phosphorylated on tyrosine residues and concomitantly activated. Both tyrosine phosphorylation and activation of p42MAPK begin within 2–3 min of fertilization, peak at approximately 15 min, then rapidly decline and disappear around the end of meiosis I. Neither the tyrosine phosphorylated form of p42MAPK nor p42MAPK activity reappears during meiosis II or the succeeding mitotic cell cycles. High doses of molybdate, a potent PTPase inhibitor, block the phosphorylation of p42MAPK and entry into the cell cycle. Lower doses of molybdate delay both p42MAPK phosphorylation and the release from cell cycle arrest, but once cells have re‐entered the cell cycle, they continue with near‐normal timing. These results argue that the transient activation of p42MAPK at fertilization is a one‐time event linked to release from cell cycle arrest. In trying to reconcile this one‐time activation of p42MAPK in clam embryos with the recurring, M‐phase specific activation of MBP/MAP kinases reported in other systems, we show that cdc2 kinase contributes a major portion of the MBP kinase activity in mitotic extracts. Furthermore, a small fraction of p42MAPK and other related kinases are present in p13suc1‐bound material, cautioning against the use of p13suc1 beads for experiments where, in addition to cdc2, the unaccounted presence of other kinase activities could be misleading.

ERKs, extracellular signal-regulated MAP-2 kinases.

A family of protein kinases, known alternatively as microtubule-associated protein-2/myelin basic protein kinases or extracellular signal-regulated kinases, is activated by numerous hormones, growth factors and other extracellular stimuli. At least two members of this family function as intermediate kinases in protein phosphorylation cascades. Their mechanisms of activation may involve autophosphorylation, which occurs on both threonine and tyrosine residues.

CNTF, FGF, and NGF collaborate to drive the terminal differentiation of MAH cells into postmitotic neurons

The differentiation of neuronal cell progenitors depends on complex interactions between intrinsic cellular programs and environmental cues. Such interactions have recently been explored using an immortalized sympathoadrenal progenitor cell line, MAH. These studies have revealed that depolarizing conditions, in combination with exposure to FGF, can induce responsiveness to NGF. Here we report that CNTF, which utilizes an intracellular signaling pathway distinct from that of both FGF and NGF, can collaborate with FGF to promote efficiently the differentiation of MAH progenitor cells to a stage remarkably reminiscent of NGF-dependent, postmitotic sympathetic neurons. We also find that similar collaborative interactions can occur during transdifferentiation of normal cultured chromaffin cells into sympathetic neurons.

ERK3 Is a Constitutively Nuclear Protein Kinase

The ERK3 cDNA predicts a protein of 62,000 in size with a C-terminal domain that extends 180 amino acids beyond the conserved core of ERK family protein kinases. Immunoblotting with antibodies raised to recombinant protein and to peptides from the catalytic core and three regions of the C-terminal tail revealed that ERK3 is the expected size and is ubiquitously expressed in a variety of cell lines and tissues. ERK3, unlike the MAP kinases ERK1 and ERK2, is localized in the nucleus in exponentially growing, quiescent, and growth factor-stimulated cells. If the 180 amino acids at its C terminus are deleted, the resulting ERK3 fragment of 45 kDa is still found primarily in the nucleus, indicating that the C terminus is not required for its localization. Recombinant ERK3 expressed in mammalian cells or in bacteria is a protein kinase, as deduced from its capacity to autophosphorylate. Mutation of a conserved residue (Asp) expected to be involved in catalysis eliminated autophosphorylation. Ser of ERK3, which corresponds to Thr, one of the activating phosphorylation sites of ERK2, is autophosphorylated in vitro and phosphorylated in vivo. Despite marked similarities to ERK1 and ERK2, ERK3 does not phosphorylate typical MAP kinase substrates, indicating that it has distinct functions.

Extracellular signal-regulated kinases in T cells: Characterization of human ERK1 and ERK2 cDNAs☆☆☆

Extracellular signal-regulated kinases 1 and 2 are growth factor-sensitive serine/threonine kinases. cDNAs for both human kinases were isolated and sequenced. The nucleic acid and deduced protein sequences of human extracellular signal-regulated kinase 1 were 88% and 96% identical, respectively, to the homologous rat sequences. The nucleic acid and deduced protein sequences of human extracellular signal-regulated kinase 2 were 90% and 98% identical, respectively, to the corresponding rat sequences. A human extracellular signal-regulated kinase 2 specific probe was used to demonstrate that the mRNA for this kinase was present in T cells and did not change with activation. The deduced protein sequences of both human kinases were greater than 95% identical to two Xenopus kinase sequences, indicating that these enzymes are highly conserved across species.

Neurotrophic factors, their receptors, and the signal transduction pathways they activate

Our studies of the spatiotemporal availability of neurotrophic factors, coupled with tagged ligand binding assays that identify cell bearing receptors for these factors, should lead toward defining the physiological roles of these molecules in the animal. The use of the tagged ligands to identify factor-responsive cell lines has also provided new model systems for the examination of ligand-receptor interactions, as well as for the study of the subsequent induction of intracellular response pathways. To obtain insights into such intracellular pathways, we have molecularly cloned genes encoding a family of serine-threonine protein kinases, most closely related to kinases involved in the yeast response to pheromones. These kinases may be crucial regulators of early steps in the response of mammalian cells to neurotrophic factors as well as other extracellular signals.