Hydrophobic as well as charged residues in both MEK1 and ERK2 are important for their proper docking.

Docking between MEK1 and ERK2 is required for their stable interaction and efficient signal transmission. The MEK1 N terminus contains the ERK docking or D domain that consists of conserved hydrophobic and basic residues. We mutated the hydrophobic and basic residues individually and found that loss of either type reduced MEK1 phosphorylation of ERK2 in vitro and its ability to bind to ERK2 in vivo. Moreover, ERK2 was localized in both the cytoplasm and the nucleus when co-expressed with MEK1 that had mutations in either the hydrophobic or the basic residues. We then identified two conserved hydrophobic residues on ERK2 that play roles in docking with MEK1. Mutating these residues to alanine reduced the interaction of ERK2 with MEK1 in cells. These mutations also reduced the phosphorylation of MEK1 by ERK2 but had little effect on phosphorylation of MBP by ERK2. Finally, we generated docking site mutants in ERK2-MEK1 fusion proteins. Although the mutation of the MEK1 D domain significantly reduced ERK2-MEK1 activity, mutations of the putatively complementary acidic residues and hydrophobic residues on ERK2 did not change its activity. However, both types of mutations decreased the phosphorylation of Elk-1 caused by ERK2-MEK1 fusion proteins. These findings suggest complex interactions of MEK1 D domains with ERK2 that influence its activation and its effects on substrates.

WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II.

We have cloned and characterized a novel mammalian serine/threonine protein kinase WNK1 (with no lysine (K)) from a rat brain cDNA library. WNK1 has 2126 amino acids and can be detected as a protein of approximately 230 kDa in various cell lines and rat tissues. WNK1 contains a small N-terminal domain followed by the kinase domain and a long C-terminal tail. The WNK1 kinase domain has the greatest similarity to the MEKK protein kinase family. However, overexpression of WNK1 in HEK293 cells exerts no detectable effect on the activity of known, co-transfected mitogen-activated protein kinases, suggesting that it belongs to a distinct pathway. WNK1 phosphorylates the exogenous substrate myelin basic protein as well as itself mostly on serine residues, confirming that it is a serine/threonine protein kinase. The demonstration of activity was striking because WNK1, and its homologs in other organisms lack the invariant catalytic lysine in subdomain II of protein kinases that is crucial for binding to ATP. A model of WNK1 using the structure of cAMP-dependent protein kinase suggests that lysine 233 in kinase subdomain I may provide this function. Mutation of this lysine residue to methionine eliminates WNK1 activity, consistent with the conclusion that it is required for catalysis. This distinct organization of catalytic residues indicates that WNK1 belongs to a novel family of serine/threonine protein kinases.

Stimulation of NFkappa B activity by multiple signaling pathways requires PAK1.

The p21-activated kinase (PAK1) is a serine-threonine protein kinase that is activated by binding to the Rho family small G proteins Rac and Cdc42hs. Both Rac and Cdc42hs have been shown to regulate the activity of the transcription factor NFkappaB. Here we show that expression of active Ras, Raf-1, or Rac1 in fibroblasts stimulates NFkappaB in a PAK1-dependent manner and that expression of active PAK1 can stimulate NFkappaB on its own. Similarly, in macrophages activation of NFkappaB as well as transcription from the tumor necrosis factor alpha promoter depends on PAK1. In these cells lipopolysaccharide is a potent activator of PAK1 kinase activity. We also demonstrate that expression of active PAK1 stimulates the nuclear translocation of the p65 subunit of NFkappaB but does not activate the inhibitor of kappaB kinases alpha or beta. These data demonstrate that PAK1 is a crucial signaling molecule involved in NFkappaB activation by multiple stimuli.

A constitutively active and nuclear form of the MAP kinase ERK2 is sufficient for neurite outgrowth and cell transformation.

BACKGROUND: Mitogen-activated protein (MAP) kinases are ubiquitous components of many signal transduction pathways. Constitutively active variants have been isolated for every component of the extracellular-signal-regulated kinase 1 (ERK1) and ERK2 MAP kinase pathway except for the ERK itself. RESULTS: To create an activated ERK2 variant, we fused ERK2 to the low activity form of its upstream regulator, the MAP kinase kinase MEK1. The ERK2 in this fusion protein was active in the absence of extracellular signals. Expression of the fusion protein in mammalian cells did not activate endogenous ERK1 or ERK2. It was sufficient, however, to induce activation of the transcription factors Elk-1 and AP-1, neurite extension in PC12 cells in the absence of nerve growth factor, and foci of morphologically and growth-transformed NIH3T3 cells, if the fusion protein was localized to the nucleus. A cytoplasmic fusion protein was without effect. CONCLUSIONS: Activation of ERK2 is sufficient to cause several transcriptional and phenotypic responses in mammalian cells. Nuclear localization of activated ERK2 is required to induce these events.

Differential effects of PAK1-activating mutations reveal activity-dependent and -independent effects on cytoskeletal regulation.

PAKs are serine/threonine protein kinases that are activated by binding to Rac or Cdc42hs. Different forms of activated PAK1 have been reported to either promote membrane ruffling and focal adhesion assembly or cause focal adhesion disassembly and stress fiber dissolution. To understand the basis for these distinct morphological effects, we have examined the mechanism of mutational activation of PAK1, and characterized the effects of different active PAK1 proteins on cytoskeletal structure in vivo. We find that PAK1 contains an autoinhibitory domain that overlaps with its small G protein binding domain and that two separate activating mutations within this regulatory region each decrease autoinhibitory activity. Because only one of these mutations affects Cdc42hs binding activity, this indicates that activation of PAK1 by these mutations results from interference with the function of the autoinhibitory domain and not with small G protein binding activity. When we examined the morphological effects of these different forms of PAK1 in vivo, we found that PAK1 kinase activity was associated with disassembly of focal adhesions and actin stress fibers and that this may require interaction with potential SH3 domain-containing proteins. Lamellipodia formation and membrane ruffling caused by active PAK1 expression, however, was independent of PAK1 catalytic activity and likely requires interaction among multiple proteins binding to the PAK1 regulatory domain.