A Two‐Dimensional Electrophoresis Study of Phosphorylation and Dephosphorylation of Chromaffin Cell Proteins in Response to a Secretory Stimulus

Abstract

Phosphorylated proteins of bovine chromaffin cells, radioactively labeled with [32P]orthophosphate, have been analyzed by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Complex two-dimensional electrophoretograms were studied with the aid of computer-assisted image analysis (CAIA). A database map of 32P-labeled proteins was constructed; approximately 500 polypeptides have been detected, numbered, and characterized according to the intensity of labeling, molecular weight, and isoelectric point. The database was constructed from cells kept in resting conditions or stimulated with 59 mM K+ in 2.5 mM Ca2+ or in 0 Ca2+ solution. These manipulations caused statistically significant changes in the degree of phosphorylation of 20 proteins; they were classified as Ca2+-dependent substrates for the phosphorylation or dephosphorylation processes. These changes were also shown in cells stimulated in the presence of the Ca2+ channel activator Bay K 8644. New proteins that show as much as a fivefold increase in their phosphorylation state during cell stimulation have been located with this methodology, as well as many others that had not previously been detected with conventional methods. These experiments provide the first CAIA database of chromaffin cell phosphoproteins; the map constructed with these data will allow the location of specific phosphoproteins and serve as a reference for future ongoing studies. The database will continue to grow to identify more proteins and to facilitate the comparison of complex patterns obtained in different laboratories for normal and transformed pheochromocytoma PC12 cells.

Stimulus-induced association of Ca(2+)-binding proteins with the plasma membrane detected in situ by photolabeling of intact chromaffin and PC12 cells.

“…Another strongly INA-labeled band is the one at 28 kDa (pI 5.3). C6td et al (13) observed high stimulus-induced phosphorylation in a protein running at 29 kDa (pI 5.7 and 5.9) and a band with essentially identical characteristics was found by Gutierrez et al (32). Similar situations obtain for our 34-(pI 4.8), 38-(pI 5.4), and 42-(pI 4.8-6.3) kDa components, all undergoing strong stimulus-induced INA labeling, for all of which there exists a close parallelism with values cited in the literature (13,27,32).…”

“…C6td et al (13) observed high stimulus-induced phosphorylation in a protein running at 29 kDa (pI 5.7 and 5.9) and a band with essentially identical characteristics was found by Gutierrez et al (32). Similar situations obtain for our 34-(pI 4.8), 38-(pI 5.4), and 42-(pI 4.8-6.3) kDa components, all undergoing strong stimulus-induced INA labeling, for all of which there exists a close parallelism with values cited in the literature (13,27,32). While the nature of most of these proteins is unknown at present, this apparent correlation between phosphorylation and close interaction with the plasma membrane perhaps gives us an initial clue regarding at least one aspect of the exocytotic mechanism.…”

The distribution of mitochondria and endoplasmic reticulum in relation with secretory sites in chromaffin cells

“…Chromaffin cells were isolated from bovine adrenal glands by collagenase digestion, and they were further separated from the debris and erythrocytes by centrifugation on Percoll gradients as described previously (Gutierrez et al, 1988;Gil et al, 2002). The cells were maintained as monolayer cultures in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 10 mM cytosine arabinoside, 10 mM 5-fluoro-29-deoxyuridine, 50 U/ml penicillin and 50 mg/ml streptomycin.…”

New Roles of Myosin II during Vesicle Transport and Fusion in Chromaffin Cells

“…A number of observations suggest that conventional myosin II activity influences catecholamine secretion. For example, chromaffin cell stimulation induces the calcium-dependent phosphorylation of the regulatory subunit of myosin light chain (RLC) 1 (5)(6)(7), and as such, constitutes the basic mechanism for governing the interaction of non-muscular myosin II with actin (8). In addition, phosphorylation of non-muscle myosins by the specific enzyme myosin light chain kinase (MLCK), may be essential to this proteins role in secretion, because inhibition of MLCK by a variety of chemicals abrogates secretion in permeabilized (9 -12) or intact chromaffin cells (2).…”