Jean de Gunzburg

The cAMP-dependent protein kinase phosphorylates the rap1 protein in vitro as well as in intact fibroblasts, but not the closely related rap2 protein.

The products of rap genes (rap1A, rap1B and rap2) are small molecular weight GTP-binding proteins that share approximately 50% homology with ras-p21s. It had previously been shown that a rap1 protein (also named Krev-1 or smg p21) could be phosphorylated on serine residues by the cAMP-dependent protein kinase (PKA) in vitro as well as in intact platelets stimulated by prostaglandin E1. We show here that the rap1A protein purified from recombinant bacteria is phosphorylated in vitro by the catalytic subunit of PKA and that the deletion of the 17 C-terminal amino acids leads to the loss of this phosphorylation. This suggests that the serine residue at position 180 constitutes the site of phosphorylation of the rap1A protein by PKA. The rap1 protein can also be phosphorylated by PKA in intact fibroblasts; this phenomenon is independent of their proliferative state. In contrast, protein kinase C (PKC) does not phosphorylate the rap1 proteins, neither in vitro nor in vivo. Finally, the 60% homologous rap2 protein is neither phosphorylated in vitro nor in vivo by PKA or PKC.

Dynamic changes in Rap1 activity are required for cell retraction and spreading during mitosis.

At the onset of mitosis, most adherent cells undergo cell retraction characterised by the disassembly of focal adhesions and actin stress fibres. Mitosis takes place in rounded cells, and the two daughter cells spread again after cytokinesis. Because of the well-documented ability of the small GTPase Rap1 to stimulate integrin-dependent adhesion and spreading, we assessed its role during mitosis. We show that Rap1 activity is regulated during this process. Changes in Rap1 activity play an essential role in regulating cell retraction and spreading, respectively, before and after mitosis of HeLa cells. Indeed, endogenous Rap1 is inhibited at the onset of mitosis; conversely, constitutive activation of Rap1 inhibits the disassembly of premitotic focal adhesions and of the actin cytoskeleton, leading to delayed mitosis and to cytokinesis defects. Rap1 activity slowly increases after mitosis ends; inhibition of Rap1 activation by the ectopic expression of the dominant-negative Rap1[S17A] mutant prevents the rounded cells from spreading after mitosis. For the first time, we provide evidence for the direct regulation of adhesion processes during mitosis via the activity of the Rap1 GTPase.

Biochemical and Structural Characterization of the Gem GTPase

RGK proteins, encompassing Rad, Gem, Rem1, and Rem2, constitute an intriguing branch of the Ras superfamily; their expression is regulated at the transcription level, they exhibit atypical nucleotide binding motifs, and they carry both large N- and C-terminal extensions. Biochemical and structural studies are required to better understand how such proteins function. Here, we report the first structure for a RGK protein: the crystal structure of a truncated form of the human Gem protein (G domain plus the first part of the C-terminal extension) in complex with Mg·GDP at 2.1 Å resolution. It reveals that the G-domain fold and Mg·GDP binding site of Gem are similar to those found for other Ras family GTPases. The first part of the C-terminal extension adopts an α-helical conformation that extends along the α5 helix and interacts with the tip of the interswitch. Biochemical studies show that the affinities of Gem for GDP and GTP are considerably lower (micromolar range) compared with H-Ras, independent of the presence or absence of N- and C-terminal extensions, whereas its GTPase activity is higher than that of H-Ras and regulated by both extensions. We show how the bulky DXWEX motif, characteristic of the switch II of RGK proteins, affects the conformation of switch I and the phosphate-binding site. Altogether, our data reveal that Gem is a bona fide GTPase that exhibits striking structural and biochemical features that should impact its regulation and cellular activities.

Ultrastructural localization of the small GTP-binding protein Rap1 in human platelets and megakaryocytes

Summary. Several functions have been proposed for Rap 1B in human platelets, including the regulation of phospholipase (PL) Cγ and Ca2+ ATPase. However, its localization is largely unknown. In the present study we have investigated the subcellular distribution of Rap1 by immunocytochemical techniques using affinity purified polyclonal antibodies raised against residues 121–137 common to the 95% homologous Rap1A and Rap1B proteins. By immuno‐fluorescence, a positive labelling was obtained on intact resting platelets and was abolished after adsorption of the antibodies with the control peptide. Immunoelectron microscopy was then used to further define the subcellular localization of Rap1B in platelets and megakaryocytes (MK). In resting cells, immunolabelling for Rap1B was associated with the plasma membrane, mostly at its inner face, and lined the membrane of the open canalicular system (OCS). Some labelling was also found outlining the α‐granules, identified as such by a double labelling with an anti‐GPIIb‐IIIa. On thrombasthenic platelets the same localization was observed. When platelets were stimulated by thrombin, immunolabelling for Rap1B was redistributed to the zones of fusion of the granules with the OCS, and to the plasma membrane with a higher concentration on pseudopods. Human MK expressed Rap1 and the staining revealed the association of the protein with the demarcation membranes and α‐granules.

Novel Rap1 dominant-negative mutants interfere selectively with C3G and Epac

Rap1 is a Ras-related GTPase that is principally involved in integrin- and E-cadherin-mediated adhesion. Rap1 is transiently activated in response to many incoming signals via a large family of guanine nucleotide exchange factors (GEFs). The lack of potent Rap1 dominant-negative mutants has limited our ability to decipher Rap1-dependent pathways; we have therefore developed a procedure to generate such mutants consisting in the oligonucleotide-mediated mutagenesis of residues 14–19, selection of mutants presenting an enhanced interaction with Epac2 by yeast two-hybrid screening and counter-screening for mutants still interacting with Rap effectors. In detail analysis of their interaction capacity with various Rap-GEFs in the yeast two-hybrid system revealed that mutants of residues 15 and 16 interacted with Epacs, C3G and CalDAG-GEFI, whereas mutants of position 17 had selectively lost their ability to bind CalDAG-GEFI as well as, for some, C3G. In cellular models where Rap1 is activated via endogenous GEFs, the Rap1[S17A] mutant inhibits both the cAMP–Epac and EGF–C3G pathways, whereas Rap1[G15D] selectively interferes with the latter. Finally, Rap1[S17A] is able to act as a bona fide dominant-negative mutant in vivo since it phenocopies the eye-reducing and lethal effects of D-Rap1 deficiency in Drosophila, effects that are overcome by the overexpression of D-Epac or D-Rap1.

Regulation of the GTPase activity of the ras-related rap2 protein

The small GTP-binding protein rap2A exhibits a high level of identity with rap1 and ras proteins (60% and 46%, respectively). Nevertheless, its intrinsic GTPase activity is not stimulated by ras-GAP, and unlike the rap1A protein, it cannot compete with ras proteins for their interaction with ras-GAP. In addition, rap1-GAPm that is highly active on the GTPase activity of the rap1A product, also stimulates the GTPase activity of the rap2A protein but with a 30-40-fold lower efficiency. An activity that greatly stimulated the GTPase activity of the rap2 protein (rap2-GAP) was found in bovine brain cytosol and purified. However, it copurified with the cytosolic form of rap1-GAP and was more efficient at stimulating the GTPase activity of the rap1 protein; this 55 kD polypeptide, that is recognized by an antibody raised against rap1-GAPm, likely represents a degraded and soluble form of the full size 89 kD molecule. In bovine brain membranes, a weak GAP activity toward the rap2A protein was also detected; however, it was also attributable to the membrane-associated rap1-GAPm. Thus, it appears that a single rap-GAP protein, complete or degraded, is able to stimulate the GTPase activity of both rap1 and rap2 proteins.

Abnormal cAMP-induced phosphorylation of rap 1 protein in grey platelet syndrome platelets

Summary We previously demonstrated abnormal Ca2+ transport by microsomes in platelets from a grey platelet syndrome patient. Here, we investigated the platelet Ca2+ ATPases that mediate this transport, as well as its possible regulation by rap 1 protein. We showed that grey platelet syndrome platelets expressed the same two distinct Ca2+ ATPases as those recently described in normal platelets; the 100 kD SERCA2‐b isoform (Sarco/Endoplasmic Reticulum Ca2+ATPase) and a new 97 kD SERCA isoform. The two Ca2+ ATPases formed similar amounts of transient phosphorylated intermediates. The expression of these two Ca2+ ATPases was compared by Western blotting using specific antibodies, which again emerged in similar amounts in normal and grey platelet syndrome platelets. As regards the protein phosphorylated by cAMP, it was found to be identical to rap 1 protein when it was immunoprecipitated with an antibody raised against a synthetic peptide specific for rap 1 protein. Although the expression of rap 1 protein was similar in membranes isolated from grey platelet syndrome and normal platelets, its exogenous phosphorylation by cAMP was abnormal, with a concentration (10 μg/ml) of the catalytic subunits of the cAMP‐dependent protein kinase (C.Sub.), as it decreased to half the control level.

Phosphorylation of rap Proteins by the cAMP-Dependent Protein Kinase

The products of rap genes (rap1A, rap1B, rap2A, rap2B) are small molecular weight GTP-binding proteins that exhibit striking similarities with ras p21s1–4. In particular, ras and rap proteins share a conserved “effector” region spanning residues 32–42 through which ras-p21s are thought to exert their biological effects; they also have a C-terminal CAAX sequence (where A is an aliphatic residue and X any amino acid) responsible for posttranslational modification and membrane binding of ras proteins5. The identity of the “effector” domain between ras and rap proteins had suggested that rap proteins could antagonize the activity of ras proteins by competing for a common effector. Independently, M. Noda’s group isolated a cDNA, Krev-1, whose overexpression could revert the transformed phenotype of Kirsten sarcoma-virus transformed NIH 3T3 cells6; the sequence of the Krev-1 protein was identical to that of the rap1A protein. Moreover, in vitro, the rap1A protein has been shown to be able to compete efficiently with ras p21 for interaction with GAP7(GTPase Activating protein), which may constitute the effector of ras p218–12.

Identification of a Protein Interacting with ras-p21- by Chemical Cross-Linking

There is accumulating evidence showing that ras oncogenes (Ha-ras, K-ras and N-ras) are involved in the processes of oncogenesis and cellular transformation1 . These genes encode highly homologous proteins of molecular weight 21 ,000 (p21) that are found in all mammalian tissues and are very conserved throughout evolution. ras-p21s are bound to the inner surface of the plasma membrane ; they bind GTP and GDP, and exhibit an intrinsic GTPase activity. A cytoplasmic protein of MW 110–120,000 has recently been identified by its capacity to enhance 100–500 fold the GTPase activity of ras-p21s (GAP) 2 ; it has been shown to interact with a domain of p21s necessary for their biological activity (amino acids 30–42 called “effector domain”) and may therefore constitute an effector of their physiological action3–6 . Point mutations resulting in the change of amino acids 12, 13, 59 or 61 have been shown to “activate” the oncogenic potential of ras oncogenes leading to cellular transformation. The GTPase activity of such mutant proteins is no longer activated by GAP, blocking them in a GTP-bound state that is thought to be the active form of p21s.