Ronald E. Gates

Calcium facilitates endogenous proteolysis of the EGF receptor-kinase

Abstract An intracellular endogenous protease requiring 1 mM Ca 2+ for activity converts the 180K dalton form of the phosphorylatable epidermal growth factor receptor-kinase to the 160K dalton form. In A-431 human epidermoid carcinoma cells this protease is: (1) activated by breaking cells in the presence of Ca 2+ , (2) inhibited by leupeptin, (3) active at neutral and slightly alkaline pH, and (4) soluble. These properties identify this endogenous protease as the widely distributed calcium-activated neutral protease.

Proteolysis of the epidermal growth factor receptor by endogenous calcium-activated neutral protease from rat liver

In A-431 membranes but not in rat liver membranes, the epidermal growth factor (EGF) receptor was converted from a Mr=180,000 to a Mr=160,000 form by a protease activated when cells were broken in the presence of calcium. Calcium-activated neutral protease (CANP) activity in rat liver cytosol was separated from its protein inhibitor by DEAE-cellulose chromatography. When fractions containing this protease activity were incubated with rat liver membranes in the presence of calcium, the Mr=180,000 form of the receptor was converted to the Mr=160,000 form. This conversion was blocked both by the separated endogenous inhibitor and by leupeptin. Apparently CANP is a highly regulated endogenous protease which could degrade the EGF receptor-kinase in most tissues.

Calcium-activated neutral protease purified from beef lung: Properties and use in defining structure of epidermal growth factor receptors

Ca2+-Requiring proteases degrade cytosolic and integral membrane proteins as well as alter, by limited proteolysis, the activity of certain protein kinases. When cells are lysed, a Ca2+-requiring protease degrades the epidermal growth factor (EGF) receptor, an integral membrane protein with an intrinsic kinase activity, from its 170-kDa form to a 150-kDa form. This Ca2+-requiring protease has all of the characteristics of calcium-activated neutral protease (CANP). To show that CANP is the protease uniquely responsible for the degradation of the native EGF receptor in vitro, CANP was highly purified from beef lung. This affinity purified CANP had properties previously described for other CANPs: heterodimer of 80 and 30 kDa; neutral pH optimum; activation by millimolar Ca2+; and inhibition by an endogenous, heat-stable proteinaceous inhibitor, by leupeptin, and by sulfhydryl alkylating agents. Using the EGF receptor labeled by covalent attachment to 125I-EGF, this purified CANP quantitatively generated the 150-kDa form from the native receptor in A-431 cell membranes. As with the native receptor, the 150-kDa receptor forms produced by the endogenous Ca2+-requiring protease, by CANP, by chymotrypsin, and by elastase were all capable of EGF-stimulated autophosphorylation. When the 150-kDa receptor forms were generated by the three exogenously added proteases, autophosphorylation with [gamma-32P]ATP followed by trypsinization produced 32P-labeled peptides that were not the same. However, the tryptic 32P-labeled peptides from the autophosphorylated 150-kDa receptor form produced by CANP or by the endogenous Ca2+-requiring protease were identical. These data indicate that CANP is identical to the endogenous Ca2+-requiring protease responsible for producing the autophosphorylating 150-kDa receptor form from the native EGF receptor when cells are lysed.

Ectoprotein kinase activity of the isolated rat adipocyte

Intact adipocytes exhibit ectoprotein kinase activity as reflected by their ability to catalyze the transfer of the terminal phosphate of (γ-32P) ATP to histone added to a cell suspension. This activity is substrate, time and cell number dependent. Lineweaver-Burk plots gave Km and Vmax values for ATP of 5 × 10−5 M and 7.14 pmoles/min/1.5 × 105 cells. Cyclic AMP but not cyclic GMP in μM concentrations stimulates ectoprotein kinase activity. The controlled tryptic digestion of intact cells results in reduction of ectoprotein kinase activity. This activity is not due to leakage of intracellular protein kinases during the preparative procedure nor to penetration of histone into the cells. Additional phosphoproteins not accessible to endogenous protein kinase activity are also localized on the external surface of the intact fat cell.

Altered distribution and expression of protein tyrosine phosphatases in normal human skin as compared to squamous cell carcinomas

Amounts and subcellular localizations of 4 protein tyrosine phosphatases (PTPs) were compared in cultured normal human keratinocytes, an immortalized keratinocyte cell line, and 2 squamous cell carcinoma (SCC) lines. Cellular localizations for PTPs were determined in biopsies of normal human skin and SCCs. Compared to normal keratinocytes, SCC cell lines had higher levels of PTP‐1B and T‐cell PTP and comparable levels of PTP‐1C or PTP‐1D. The subcellular localization of each PTP was similar in the 3 types of keratinocytes with PTP‐1B localizing to the endoplasmic reticulum, T‐cell PTP exclusively found in the nucleus, PTP‐1C localized to the plasma membrane, cytosol and nucleus, and PTP‐1D present in both cytosol and nucleus. Compared to normal skin, immunoreactive PTP‐1B was markedly increased in the invasive margins of SCCs while T‐cell PTP was generally increased in tumors. PTP‐1C immunostaining varied between cells with no obvious difference between normal and neoplastic tissues. The intensity and distribution of immunoreactive PTP‐1D varied greatly between cells within tumors. These differences in amounts and in cellular and subcellular localization of these PTPs, especially those differences in invasive margins of SCCs, may reflect the diverse roles these PTPs play in the proliferation and invasive potential of neoplastic keratinocytes.

Elimination of interfering substances in the presence of detergent in the bicinchoninic acid protein assay

Substantial time and effort are necessary to eliminate interference due to thiols and reducing sugars in the bicinchoninic acid protein assay when detergents such as sodium dodecyl sulfate (SDS) are present. A method to eliminate these interfering substances based on the binding of proteins to positively charged nylon membranes at pH 8.5 followed by methanol and aqueous washes is described. Color development using bicinchoninic acid occurs in solution with only a minor contribution from the nylon membrane itself. The retention of most proteins on the membrane is nearly quantitative and is only slightly decreased by high salt or detergent in the assay mixture. The interference due to thiol compounds such as beta-mercaptoethanol and dithiothreitol is completely eliminated while that due to reducing sugars such as glucose is substantially reduced. Therefore, the amount of protein dissolved in the sample buffer used for SDS-polyacrylamide gel electrophoresis (2% SDS and 5% beta-mercaptoethanol) can be determined with this bound protein assay.

A miniature molecular-sieving column assay for cytoplasmic vitamin A-binding proteins

Cytoplasmic vitamin A-binding proteins were measured by a method using centrifugation of gel-exclusion columns and compared to the sucrose gradient method. The gel-exclusion method analyzed 18 samples simultaneously on one table-top centrifuge, while the sucrose gradient method required use of three ultracentrifuges to process 18 samples simultaneously. Multiple 2-min low-speed centrifugations of test cytosol applied to miniature molecular-sieving columns was a faster (1/2 the working time for 18 samples), more convenient, and more accurate method for measuring cytoplasmic vitamin A-binding proteins than was the sucrose gradient method. Using the same cytosol and [3H]retinoid preparations, the rapid gel-exclusion method results were only slightly lower than those obtained by radioimmunoassay but 50% higher than the values obtained by the sucrose gradient assay. The methodology described may be useful not only for cytoplasmic vitamin A-binding proteins but also for other similar binding protein assays.

Altered vitamin A-binding proteins in carcinoma of the head and neck.

Since vitamin A inhibits the terminal differentiation of keratinocytes, elevated levels of cytoplasmic vitamin A‐binding proteins could explain the reduced tendency of squamous cell carcinomas to undergo terminal differentiation. The levels of cellular retinol‐binding protein (CRBP) and cellular retinoic acid‐binding protein (CRABP) were determined in 37 squamous cell carcinomas of the head and neck and were compared with the corresponding levels in adjacent normal tissue. The CRBP levels expressed per milligram of DNA were increased threefold (P = 0.0005) in the tumor as compared with normal tissue. In contrast, CRABP levels expressed per milligram were decreased 2.5‐fold (P = 0.0007) in the tumor as compared with normal tissue. Despite significant differences in retinoid‐binding protein levels between tumor and adjacent normal tissue, there was no significant correlation of these differences with any of the patient clinical parameters or any of the tumor growth characteristics. The increased CRBP levels may explain why terminal differentiation, an important mechanism for limiting growth in normal keratinocytes, is less readily induced in squamous cell carcinomas.

Alkaline hydrolysis and multiple site autophosphorylation differ for two forms of the epidermal growth factor receptor

Different tyrosines are autophosphorylated on the native and on the protease-generated 150 kDa forms of the epidermal growth factor receptor. High ATP concentrations increase the apparent molecular weight of already phosphorylated native receptors but not of the 150 kDa form, indicating that only the native receptor has multiple autophosphorylation sites available. The non-identity of the tyrosine-phosphates on the native and 150 kDa receptor forms is seen in their response to alkaline hydrolysis (10% and 40% resistant, respectively). Since the liberated phosphate is peptide bound, the native receptor fails to be alkali-resistant because of which peptide bonds are hydrolyzed.

Detergent solubilization is a prerequisite for aggregation-induced stimulation of epidermal growth factor (EGF) receptor kinase

Unlike EGF, concanavalin A and wheat germ agglutinin do not increase EGF receptor-kinase activity in intact A-431 membranes. However, they increase both autophosphorylation and phosphorylation of exogenous substrates about twice as much as EGF if the membranes are solubilized in detergent. Following solubilization, autophosphorylation due to the combined presence of a lectin and EGF is additive suggesting that each increases kinase activity by a different mechanism. These different mechanisms were studied by autophosphorylating membranes at increasing detergent concentrations after they had been permeabilized to [gamma-32P]ATP with alamethicin. As the detergent concentration increased, EGF stimulated autophosphorylation decreased 3-fold and 6-fold for the native 170 kDa receptor and for a protease-generated 150 kDa receptor form, respectively. However, in the presence of either lectin the same increase in detergent concentration only slightly altered the autophosphorylation rates which never exceeded the rate measured in the absence of EGF and detergent. Hence, the lectins increase kinase activity in solubilized membranes by preventing the adverse effects of detergent on the receptor-kinase and may not be useful models for how EGF activates its receptor.