Hormonal regulation of focal adhesions in bovine adrenocortical cells: induction of paxillin dephosphorylation by adrenocorticotropic hormone

Abstract

A study of bovine adrenocortical cell shape on adrenocorticotropic hormone (ACTH) challenge showed that the cells round up and develop arborized processes. This effect was found to be (1) specific for ACTH because angiotensin II and basic fibroblast growth factor have no effect; (2) mediated by a cAMP-dependent pathway because forskolin reproduces the effect of the hormone; (3) inhibited by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor, but unchanged by okadaic acid, a serine/threonine phosphatase inhibitor; and (4) correlated with a complete loss of focal adhesions. Biochemical studies of the focal-adhesion-associated proteins showed that pp125fak, vinculin (110 kDa) and paxillin (70 kDa) were detected in the Triton X-100-insoluble fraction from adrenocortical cells. During cell adhesion on fibronectin as substratum, two major phosphotyrosine-containing proteins of molecular masses 125 and 68 kDa were immunodetected in the same fraction. A dramatic decrease in the extent of tyrosine phosphorylation of these proteins was observed within 60 min after treatment with ACTH. No change in pp125fak tyrosine phosphorylation nor in Src activity was detected. In contrast, paxillin was found to be tyrosine-dephosphorylated in a time-dependent manner in ACTH-treated cells. Sodium orthovanadate completely prevented the effect of ACTH. These observations suggest a possible role for phosphotyrosine phosphatases in hormone-dependent cellular regulatory processes.

Protein tyrosine phosphatases are involved in LH/chorionic gonadotropin and 8Br-cAMP regulation of steroidogenesis and StAR protein levels in MA-10 Leydig cells

“…In addition, we have demonstrated that human (h) CG and 8Br-cAMP produce a rapid and transient increase in total PTP activity in MA-10 cells that involves at least two PTPs (110 kDa and 50 kDa forms), and induce tyrosine dephosphorylation of endogenous proteins (Cornejo Maciel et al 2001). Moreover, we and others have described similar effects following ACTH treatment of bovine (Vilgrain et al 1998) and rat (Paz et al 1999) zona fasciculata cells.…”

“…Given that the signaling cascade triggered by steroidogenic tropic hormones is mediated by Ser/Thr kinase activation, the studies on the role played by protein dephosphorylation were initially focused on Ser/Thr phosphatases (PPs) (Iyer et al 1988, Azhar et al 1991, Sayed et al 1998, Jones et al 2000. Several recent reports indicate that tyrosine dephosphorylation is an active component of the steroidogenic pathway (Vilgrain et at 1998, Paz et al 1999, Cornejo Maciel et al 2001, Sewer & Waterman 2002, and the present study provides evidence that PTP activity participates in the mechanism by which LH/CG and 8Br-cAMP increase StAR protein levels. This conclusion is supported by the fact that the increase in the levels of both StAR protein and mRNA caused by tropic hormone or by the second messenger, 8Br-cAMP, is partially inhibited by PAO, a PTP inhibitor.…”

Interdependence of steroidogenesis and shape changes in Y1 adrenocortical cells: studies with inhibitors of phosphoprotein phosphatases

“…On application of a stimulant of the cyclic AMPdependent protein kinase (PKA) signalling pathway, such as forskolin (FSK), cyclic AMP or adrenocorticotrophin (ACTH), the cell membrane retracts and the cell shape changes rapidly from flat and adherent to round and loosely attached (Kowal 1970, Mrotek & Hall 1975, Betz & Hall 1987. Similar morphological responses to stimulation with cyclic AMP have been found in other steroidogenic cell types, including bovine, rat and human adrenal cells (Rainey et al 1983, Vilgrain et al 1998, testicular Leydig and Sertoli cells (Hall et al 1979, Spruill et al 1981, Bilinska et al 1997 and ovarian granulosa cells (Ben-Ze'ev et al 1987, Aharoni et al 1993.…”

“…Cell rounding is associated with the dephosphorylation of paxillin and involves the disassembly of focal adhesions, dissolution of stress fibres and rearrangement of the cytoskeleton. In recent studies using Y1 and bovine adrenocortical cells, activation of PKA was found to cause the selective dephosphorylation of paxillin on tyrosine residues before any detectable change in cell shape occurred, and both events were blocked by inhibitors of protein tyrosine phosphatase activity, such as sodium orthovanadate and pervanadate (Han & Rubin 1996, Vilgrain et al 1998. Recent evidence has identified one of the PTPs activated by ACTH in bovine adrenocortical cells as the cytosolic phosphatase, SHP2, suggesting that this enzyme plays a part in mediating the effects of stimulation (Rocchi et al 2000).…”

“…This change precedes the steroidogenic response and it has been suggested that the reorganisation of the actin skeleton that occurs facilitates delivery of cholesterol to the outer mitochondrial membrane thus leading to increased steroid synthesis (Betz & Hall 1987, Shiver et al 1992, Feuilloley & Vaudry 1996, Hall 1997. Recently, it has been demonstrated that an early effect of PKA activation is targeted to the protein paxillin, a component of focal adhesions which is dephosphorylated on tyrosine residues within minutes of exposure of Y1 cells and bovine adrenocortical cells to cyclic AMP (Han & Rubin 1996, Vilgrain et al 1998.…”