14-686 Sigma-AldrichLOK Protein, active, 10 µg

The Ste20 family of serine/threonine kinases plays an important role in numerous cellular functions such as growth, apoptosis, and morphogenesis. We have identified a previously cloned but uncharacterized family member termed Stk10, which is a human homolog of murine Lok, a serine/threonine kinase highly expressed in lymphocytes. Northern analysis demonstrated that the Stk10 transcript is present in many tissues, although highest expression levels are seen in hematopoietic cells. Due to close sequence homology to human Slk and Xenopus laevis xPlkk1, two polo-like kinase kinases, we investigated whether Stk10 might also play a role as a Plk1 activator. Plk1 has been shown to be overexpressed in multiple tumor types, thus attracting high interest to its potential upstream regulators. We show here that Stk10 can associate with Plk1 in cells and furthermore can phosphorylate Plk1 in vitro. Engineered NIH-3T3 cell lines that overexpress a dominant negative version of Stk10 display an altered cell cycle phenotype characterized by increased DNA content, raising the possibility that expression of a dominant negative Stk10 may impinge upon Plk1 function in vivo; it has previously been shown that unregulated expression of Plk1 can result in a variety of nuclear defects. We suggest, therefore, that Stk10 is a novel polo-like kinase kinase that cooperates with hSlk to regulate Plk1 function in human cells.

T-cell activation requires signals from both the T-cell receptor (TcR) and other co-stimulatory molecules such as CD28. TcR- and CD28-mediated signals are integrated during T-cell activation resulting in the expression of cytokine genes such as interleukin-2 (IL-2). An enhancer element (CD28RE) of the IL-2 gene specifically responsive to CD28 signals has been previously identified and characterized. This response element and an adjacent Activated Protein-1 (nuclear factor-interleukin-2B) site together (RE/AP1) were shown to complex with c-rel, AP-1 and other factors. However, details of the signal transduction pathways leading from CD28 to the composite response element remain poorly understood. We present data showing that overexpression of the serine threonine kinase, mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase kinase-1 (MEKK1), but not nuclear factor-kappa B inducing kinase, or MAP kinase/ERK kinase-1 (MEK1), can significantly increase the level of CD28RE/AP1-driven luciferase (Luc) reporter gene expression in Jurkat E6-1 cells. A MEKK1 dominant negative mutant blocked such activation induced by stimulation with Raji B cells and the superantigen staphylococcus enterotoxin E (SEE), as well as via CD3/CD28. Mutations in either site of the RE/AP1 element abolished MEKK1-induced Luc expression. Calcineurin inhibitors, CsA and FK520, or inhibitors of p38 kinase (SB 203580), or MEK1 (PD 098059), did not affect MEKK1-induced reporter activation. These results directly implicate MEKK1 in the CD28 signalling pathway that activates the CD28 response element. Co-expression of the lymphocyte-oriented kinase (LOK) kinase attenuated Raji/SEE-induced IL-2 production in Jurkat cells, as well as MEKK1 and Raji/SEE-induced reporter gene activation. These data suggest that MEKK1 and LOK may have opposing roles in regulating the CD28RE/AP1 element.

Lymphocyte-oriented kinase (LOK) is a member of the STE20/p21-activated kinase (PAK) family and expressed predominantly in lymphoid organs. Generation of LOK-deficient mice revealed that the leukocyte-function-associated antigen (LFA-1)/intercellular adhesion molecules (ICAM)-mediated aggregation of mitogen-stimulated T cells was greatly enhanced in the absence of LOK. Though levels of total LFA-1 and ICAMs as well as the active form of LFA-1 on T cell blasts were comparable in the presence and absence of LOK, clustering of active LFA-1 detected by binding of soluble ICAM-1 was accelerated in the absence of LOK. These results suggest that LOK is potentially involved in the regulation of LFA-1-mediated lymphocyte adhesion.

LOK is a new and unique member of the STE20 family with serine/threonine kinase activity, and its expression is restricted mostly to lymphoid cells in mice. We cloned the cDNA encoding the human homologue of LOK. The amino acid sequence deduced from the cDNA shows a high similarity to that of mouse LOK, with 88% identity as a whole. The kinase domains at the N-terminus and the coiled-coil regions at the C-terminus are particularly conserved, showing 98% and 93% identity, respectively. Western blot analysis with mouse LOK-specific antibody detected 130 000 Mr LOK proteins in human and rat lymphoid cell lines and tissues. The gene encoding the LOK (STK10/Stk10) gene was mapped by fluorescence in situ hybridization to chromosome 5q35.1 in human, chromosome 11A4 in mouse, and chromosome 10q12.3 in rat. By virtue of polymorphic CA repeats found in the 3' untranslated region of the mouse Stk10 gene, the Stk10 locus was further pinpointed to chromosome 11 between D11Mit53 and D11Mit84, using the intersubspecific backcross mapping panel. These results established STK10 as a new marker of human chromosome 5 to define the syntenic boundary of human chromosomes 5 and 16 on mouse chromosome 11.

We have identified a new gene, designated lok (lymphocyte-oriented kinase), that encodes a 966-amino acid protein kinase whose catalytic domain at the N terminus shows homology to that of the STE20 family members involved in mitogen-activated protein (MAP) kinase cascades. The non-catalytic domain of LOK does not have any similarity to that of other known members of the family. There is a proline-rich motif with Src homology region 3 binding potential, followed by a long coiled-coil structure at the C terminus. LOK is expressed as a 130-kDa protein, which was detected predominantly in lymphoid organs such as spleen, thymus, and bone marrow, in contrast to other mammalian members of the STE20 family. LOK phosphorylated itself as well as substrates such as myelin basic protein and histone IIA on serine and threonine residues but not on tyrosine residues, establishing LOK as a novel serine/threonine kinase. When coexpressed in COS7 cells with the known MAP kinase isoforms (ERK, JNK, and p38), LOK activated none of them in contrast to PAK- and GCK-related kinases. These results suggest that LOK could be involved in a novel signaling pathway in lymphocytes, which is distinct from the known MAP kinase cascades.