Interleukin-1 activates a novel protein kinase that phosphorylates the epidermal-growth-factor receptor peptide T669

Abstract

We have isolated from KB cells stimulated with interleukin-1 (IL-1) a protein kinase that phosphorylates a peptide (T669) based on the sequence around T669 of the epidermal growth factor (EGF) receptor. The enzyme, which had an apparent molecular mass of 45 kDa on gel-filtration chromatography, was purified 170,000-fold from cytosolic extracts by sequential chromatography on Mono Q, Mono S, phenyl-Sepharose, Superose 12, ATP-Sepharose and Mono Q. The enzyme activity co-chromatographed at the last step with a 45 kDa protein band that stained for phosphotyrosine. This peak fraction also contained some actin and a 60 kDa protein that stained weakly for phosphotyrosine. The T669 peptide is a substrate for mitogen-activated protein (MAP) kinase. Amounts of IL-1-induced T669 kinase and activated recombinant p42 MAP kinase having equal activity on T669 peptide were compared on commonly used MAP kinase substrates. T669 kinase was two or three orders of magnitude less active on myelin basic protein or microtubule-associated protein-2 than was MAP kinase. The IL-1-induced T669 kinase did not react with antiserum to p42/p44 MAP kinase. It was inactivated by treatment with protein phosphatase 2A or protein phosphotyrosine phosphatase 1B, so it may be regulated by dual phosphorylation in similar fashion to MAP kinase. The dephosphorylated enzyme was not re-activated by MAP kinase kinase. This novel enzyme could lie on a kinase cascade induced by IL-1. It may be responsible for phosphorylating T669 of the EGF receptor.

Interleukin 1 activates jun N‐terminal kinases JNK1 and JNK2 but not extracellular regulated MAP kinase (ERK) in human glomerular mesangial cells

“…3A). This chromatographic behaviour is typical for the stress-activated protein kinases as we have shown during the purification of p54 MAP kinase [7,8]. An anti-p54 MAPK-I3 antiserum, which cross-reacts with 50-and 55-kDa forms of p54 MAP-~ and 45/46-kDa jun kinases in KB epidermal carcinoma and HepG2 hepatoma cells (M. Kracht, unpublished results) recognised two major protein bands with molecular masses of about 46 and 55 kDa on SDS/PAGE in MonoQ fractions 5-13 (Fig.…”

“…All genes are highly homologous and encode proteins with a molecular mass between 46 kDa (JNK1, p54 MAP kinase y) and 5a5 kD (JNK2, p54 MAP kinase o~ and ~3) when expressed in COS cells. Recently we purified from rat liver two 50-and 55-kDa forms of IL-l-activated p54 MAP kinase a [8] and an IL-1-activated 46-kDa SAP kinase from the human epidermal cell line KB [7].…”

“…Human recombinant IL-lot was expressed in E. coli and purified as described [7]. IL-I~ was a gift from Dr. Gallati, Hoffmann LaRoche, Basel, Switzerland; IFN-~/was donated by Dr. B. Otto, Fraunhofer Institute, Hannover, Germany; IL-3, IL-4, IL-13, TNF-c~, transforming growth factor [3-1 (TGF-131) and basic fibroblast growth factor (bFGF) were all from Pharma Biotechnologie, Hannover, Germany; IL-6 was a gift from Dr. W. Fiers, Gent, Belgium; IL-8 was from Dr. 1.…”

“…The cDNA for GST p54 MAP kinase 13 was a kind gift of Dr. J.R. Woodgett (The Ontario Cancer Institute, Toronto, Ont., Canada). GST fusion proteins were expressed and purified from E. cob as glutathione S-transferase fusion proteins [8]. The mouse anti-human JNK1 antibody (clone G151-333.8) was from Pharmingen, San Diego, CA, USA.…”

“…Abbreviations: IL-I, interleukin 1; TNF, tumor necrosis factor; JNK, jun N-terminal kinase; SAPK, stress-activated protein kinase; PMA, phorbol 12-myristate 13-acetate; bFGF, basic fibroblast growth factor; PDGF, platelet-derived growth factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; M-CSF, macrophage colony-stimulating factor; LPS, lipopolysaccharide; TGF-[3, transforming growth factor [3 1 has been shown to activate various MAP kinases [6][7][8][9][10][11][12]. The aim of the present study was therefore to investigate the regulation of two major MAP kinase pathways, the jun or stressactivated protein kinases and the extracellular regulated kinases, by IL-1 in human mesangial cells.…”