RhoGDI proteins regulate the cycling and distribution of RhoGTPases (18
). Co-crystallization data of RhoGDIα bound to Cdc42 or Rac1 (38
) reveal that residues 34–57 form an ordered helix-loop-helix motif characterized by hydrophobic interactions. Acquisition of negative charge at Ser-34 may disrupt the stability of the helix-loop-helix motif, interfering with the GTPase interaction and causing release of the GTPase from RhoGDI. In addition, there are extensive interactions between GDI N termini and switch I and II regions of the GTPases. GTPase binding establishes a bridge that brings regions of the N- and C-terminal domains of RhoGDI into proximity. A striking example is Arg-66 (Cdc42), the guanidinium group of which exhibits hydrogen bonding with Asp-185 as well as Pro-30 and Ala-31 of the GDI. Additionally, hydrophobic interactions between the aliphatic portion of the Arg-66 with Gln-32 and Ile-122 of the GDI were observed (38
). Because many of the key residues of the RhoGDI N-terminal region lie immediately adjacent to Ser-34, these interactions may be disturbed by phosphorylation. However, Arg-66 of Cdc42 is highly conserved in Rac1 and RhoA, and indeed there is high sequence conservation through the switch II region, consistent with its role in GTP hydrolysis regulation, so the RhoA-specific effect of Ser-34 phosphorylation deserves further analysis.
studies identifying the site and consequences of RhoGDIα phosphorylation are supported by in vivo
studies with a Ser-34-phosphospecific antibody. Four widely differing cell types were investigated, and in each case endogenous phosphorylation of the Ser-34 residue could be detected. In MDCK cells, phorbol ester stimulates cytoskeletal reorganization and PKCα translocation to the membrane (43
), although integrin-mediated adhesion is promoted by phorbol ester in K562 cells, also associated with actin cytoskeletal reorganization (39
). In both cell types, RhoGDIα phosphorylation on Ser-34 rapidly increased in concert with these adhesion events. Additionally, and consistent with in vitro
data, RhoGDIα phosphorylation was blocked by the Gö6976 inhibitor, which has highest activity against PKCα and PKCβ1 (41
) The increase in Ser-34 phosphorylation of RhoGDIα in MDCK cells responding to phorbol ester, or HGF, is particularly interesting. Both stimulants activate RhoA rather than Rac (45
), although the molecular mechanism is unknown. The result is actin cytoskeletal reorganization and loss of cell-cell junctions. Moreover, two studies with mouse keratinocytes and KB cells showed that motility in response to these same agents was blocked both by C3 transferase and overexpression of wild-type RhoGDI (55
). These studies therefore implicated RhoGDI, with which the current data are entirely consistent. A key result is that introduction of the S34D mutant of RhoGDIα into MDCK cells activated the motility responses of MDCK cells without a need for PKC activation. In contrast, such responses were not seen in wild-type or S34A RhoGDI-expressing cells. However, we cannot rule out additional roles for guanine exchange factors and GTPase-activating proteins. Gentile et al.
) showed, for example, that HGF-promoted invasion is associated with down-regulated expression of the RacGAP, Arhgap12.
Further experiments utilized the Raichu construct as a reporter for GTP-Rho levels (33
). This sensitive indicator combined with FRET microscopy enabled the impact of RhoGDIα phosphorylation to be assessed. Expression of the S34D form of RhoGDIα in fibroblasts yielded a FRET decline (i.e.
GTP-RhoA levels increased). This provided in vivo
evidence that RhoGDIα phosphorylation regulates RhoGTP levels. In contrast, no elevation in GTP-RhoA levels was seen by transfection of wild-type RhoGDIα or an S34A form. In these cases, FRET was increased, suggesting that a form of RhoGDIα that cannot be phosphorylated sequesters GDP-RhoA. Although wild-type RhoGDIα can theoretically be phosphorylated when overexpressed, increased protein levels alter the equilibrium in favor of decreased overall phosphorylation, consistent with Takaishi et al.
Efficient Ser-34 phosphorylation of RhoGDIα by conventional PKC occurs in the presence of PtdIns(4,5)P2
, not with the often used combination of phosphatidylserine, diacylglycerol, and calcium. This inositol phospholipid can mediate PKCα activation, and a lysine-rich-binding site in the C2 domain was identified (10
). We, and others, showed that syndecan-4-mediated activation of PKCα in the presence of PtdIns(4,5)P2
was calcium-independent (13
), and this mechanism of kinase activation corresponds well to the current data showing that the PKCα/PtdIns(4,5)P2
combination efficiently phosphorylated RhoGDIα. The C2 domain of PKCα has a higher affinity for PtdIns(4,5)P2
than phosphatidylserine, so the inositide may be a kinase-targeting site (15
). Recent work shows that PKCα C2 domain may adopt a different membrane orientation when bound to PtdIns(4,5)P2
compared with phosphatidylserine (59
). In turn, this may modulate kinase activity, and our study provides some evidence that substrate specificity might be engendered this way. Therefore, we predict PKCα activation resulting from phospholipase C activity, i.e.
the generation of diacylglycerol, would not trigger RhoGDI phosphorylation and RhoA activation. By the same token, because inositol 1,4,5-trisphosphate and Ca2+
release from the endoplasmic reticulum are consequences of phospholipase C activity, the phosphorylation of RhoGDIα is presumed to be independent of calcium fluxes. Consistent with this, we find that the PtdIns(4,5)P2
-driven PKCα activation is indeed Ca2+
-independent. Moreover, RhoGDIα phosphorylation persisted in the presence of a characterized phospholipase C inhibitor, U73122 (60
Our data differ from two reports suggesting that in endothelial cells Ser-96 of RhoGDIα was subject to phosphorylation by PKCα, with a downstream increase in GTP-RhoA levels (61
) or release of RhoG (62
), a close relative of Rac1 (63
). Although the methods and cells are different, we could not demonstrate phosphorylation of Ser-96 in vitro
. A truncated form of RhoGDIα(67–204), missing the N-terminal region but containing Ser-96, was not phosphorylated by PKCα. Knezevic et al.
) utilized N-terminally green fluorescent protein-tagged RhoGDIα that may have affected the ability of PKCα to phosphorylate Ser-34. In contrast to that study, we prepared a phosphospecific antibody that revealed both the widespread occurrence of Ser-34 phosphorylation and regulation in response to PKC activation. Serine 96 lies in a consensus PKC phosphorylation site that is present in the immunoglobulin domain of mammalian RhoGDIα but not conserved across other vertebrates or isoforms. Based on structural data, the Ser-96 residue does not apparently form part of a contact site with GTPase or the hydrophobic pocket that captures the prenyl moiety. In contrast, Ser-34 is conserved across all vertebrate RhoGDIα isoforms and is present in all vertebrate β and γ isoforms for which there are data. Although Ser-34 is a major PKC phosphorylation site, in vitro
experiments suggested some residual phosphorylation after GDI mutation of Ser-34 to alanine or aspartate. The amounts varied but could represent another phosphorylation site, presumably also in the N-terminal portion of RhoGDIα.
Because RhoGDIα is an abundant cytoplasmic protein, its role is potentially significant. Our work complements that of DerMardirossian et al.
) where p21-activated kinase phosphorylation of RhoGDIα on Ser-101 and Ser-174 causes specific release of Rac1. In this case, the key sites are C-terminal and are close to each other and to the prenyl-binding cleft of the GDI (38
). It is proposed that the negative charge may destabilize this hydrophobic interaction and cause release of the Rac1. This must be a very specific effect because Rho and Cdc42 are also prenylated yet are unaffected. A second model of RhoGDI regulation involves Tyr-156 phosphorylation by Src, but only when not complexed to a GTPase (21
). This not only reduced affinity of the GDI for RhoA, Rac1, and Cdc42, but it also triggered a membrane or cortical redistribution of the protein. The current data show that Ser-34 phosphorylation can occur in free or GTPase-bound RhoGDIα. Phosphorylation on Tyr-156 was only noted in cells overexpressing a constitutively active form of Src (21
), perhaps an indicator of its transient nature. Here, Ser-34 phosphorylation was observed in several different untransfected cell types, with levels changing in response to adhesion events.
Focal adhesion and microfilament bundle assembly is promoted by GTP-RhoA (65
), with activation of the Rho kinases, with ROCK I playing a major role in primary fibroblasts (28
). Rho kinases phosphorylate myosin light chain and the myosin-binding subunit of myosin phosphatase, leading to myosin II-driven contraction. The transmembrane heparan sulfate proteoglycan, syndecan-4, supports focal adhesion assembly through the binding and persistent activation of PKCα, in the presence of PtdIns(4,5)P2
, an event upstream of GTP-RhoA (8
). In MDCK cells, Rho kinase activity is required both for focal adhesion/stress fiber formation but also lamellipodial ruffle stabilization, possibly through phosphorylation of adducin (29
). However, the upstream events that lead to the accumulation and targeting of GTP-RhoA were not known. Here, evidence suggests that a major downstream target of PKCα/PtdIns(4,5)P2
is RhoGDIα with phosphorylation on Ser-34. The result of phosphorylation is release or decreased capture of the GTPase, leading to nucleotide exchange and interaction with downstream effectors.