Protein kinase WNK2 inhibits cell proliferation by negatively modulating the activation of MEK1/ERK1/2.

The recently identified subfamily of WNK protein kinases is characterized by a unique sequence variation in the catalytic domain and four related human WNK genes were identified. Here, we describe the cloning and functional analysis of the human family member WNK2. We show that the depletion of endogenous WNK2 expression by RNA interference in human cervical HeLa cancer cells led to the activation of the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinases but, in contrast to the depletion of WNK1, had no effect on ERK5. Furthermore, expression of a kinase-dead WNK2-K207M mutant also activated ERK1/2 suggesting that WNK2 catalytic activity is required. Depletion of WNK2 expression increased G1/S progression and potentiated the cellular response to low epidermal growth factor concentrations. The molecular mechanism of ERK1/2 activation in WNK2-depleted cells lies downstream of the Raf kinases and involves MEK1 phosphorylation at serine 298 in both HeLa and HT29 colon cancer cells. This modification is linked to the upregulation of MEK1 activity toward ERK1/2. Together, these results provide evidence that WNK2 is involved in the modulation of growth factor-induced cancer cell proliferation through the MEK1/ERK1/2 pathway. The data identify WNK2 as a candidate tumor suppressor gene and suggest a coordinated activity of WNK kinases in the regulation of cell proliferation.

Cloning of a novel human Rac1b splice variant with increased expression in colorectal tumors.

Rac1 is a member of the Ras superfamily of small GTPases involved in signal transduction pathways that induce the formation of lamellipodia, stimulate cell proliferation and activate the JNK/SAPK protein kinase cascade. Here we describe that amplification by RT-PCR of the entire Rac1 coding sequence from a series of human adult and fetal tissues revealed beside the expected Rac1 cDNA, a variant product which contained additional 57 nucleotides between codons 75 and 76. This variant resulted in an in-frame insertion of 19 new amino acids immediately behind the switch II region, including two potential threonine phosphorylation sites for casein kinase II and protein kinase C. Primers designed within and downstream of the inserted nucleotide sequence allowed isolation of a genomic clone with intronic consensus sequences demonstrating that the insertion corresponds to a novel, yet undescribed exon 3b. This Rac1 splice variant, designated Rac1b, was predominantly identified in skin and epithelial tissues from the intestinal tract. Most notably, the expression of rac1b versus rac1 was found to be elevated in colorectal tumors at various stages of neoplastic progression, as compared to their respective adjacent tissues. We suggest that the 19 amino acid-insertion following the switch II region may create a novel effector binding site in rac1b, and thus participate in signaling pathways related to the normal or neoplastic growth of the intestinal mucosa.