Báo cáo Y học: Ruk is ubiquitinated but not degraded by the proteasome ppt

Specific interaction between the E3 ubiquitin ligase Cbl and all three Ruk isoforms was demonstrated by coexpression studies in Hek293 cells.. B Hek293 cells were transfected with 1.5 lg

Veterinary Sciences, the University of Edinburgh, UK; 3 Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia;

4 Institute of Molecular Biology and Genetics, Kyiv, Ukraine; 5 Institute of Cell Biology, Lviv, Ukraine

The regulator of ubiquitous kinase (Ruk) protein, also

known as CIN85 or SETA, is an adaptor-type protein

belonging to the CD2AP/CMS family It was found in

complexes with many signaling proteins, including

phos-phoinositol (PtdIns) 3-kinase (EC 2.7.1.137)

p130Cas and Crk Functional analysis of these interactions,

implicated Ruk in the regulation of apoptosis, receptor

endocytosis and cytoskeletal rearrangements We have

recently demonstrated that overexpression of Ruk induces

apoptotic death in neurons, which could be reversed by

activated forms of PtdIns 3-kinase and PKB/Akt

Further-more, Ruk was shown to be a negative regulator of PtdIns

3-kinase activity through binding to its P85 regulatory

sub-unit [Gout, I., Middleton, G., Adu, J., Ninkina, N N.,

Drobot, L B., Filonenko, V., Matsuka, G., Davies, A M.,

Waterfield, M & Buchman, V L (2000) Embo J 19, 4015–4025] Here, we report for the first time, that all three isoforms of Ruk (L, M and S) are ubiquitinated Specific interaction between the E3 ubiquitin ligase Cbl and all three Ruk isoforms was demonstrated by coexpression studies in Hek293 cells The interaction of Ruk M and S isoforms with Cbl was found to be mediated via heterodimerization with Ruk L The use of proteosomal and lysosomal inhibitors clearly indicated that ubiquitination of Ruk L does not lead

to its degradation Based on this study, we propose a possible mechanism for the regulation of Ruk function by ubiquiti-nation

Keywords: signal transduction; adaptor protein; ubiquitina-tion; proteasomal degradation

Ubiquitination of proteins plays a major role in the

regulation of various cellular processes The best studied

function of ubiquitination is its role in protein degradation,

where polyubiquitinated proteins are recognized by the 26S

proteasome or, in certain cases, by the lysosomes/vacuole

and rapidly degraded Ubiquitination of target proteins

involves a cascade of reactions catalysed by the E1, E2 and

E3 enzymes Ubiquitin (Ub) is first activated by an

activating enzyme (E1) to form a high energy thioester

bond between Ub and E1 and is then transferred to a

conjugating enzyme (E2) The Ub protein ligases or E3s are

responsible for specific substrate recognition and for

promoting covalent Ub ligation to the target protein Thus,

the E3s provide specificity to the Ub system [1,2]

The Cbl proteins form a family of related proteins

harboring several highly conserved domains, such as an

N-terminal variant SH2 domain, a RING finger and a

C-terminal proline-rich domain containing potential

tyro-sine phosphorylation sites Previous studies have shown that

c-Cbl and Cbl-b

2 function as adaptor proteins by interacting

with other signaling molecules through their various

pro-tein–protein interacting motifs [3]

Biochemical and genetic studies have shown that Cbl family proteins, including those from Drosophila and Caenorhabditis elegans, attenuate intracellular signaling induced by the engagement of cell surface receptors The mechanism underlying the negative regulation of activated receptors by Cbl proteins has been recently described Cbl functions as an E3 Ub protein ligase, which mediates the ubiquitination of activated receptor tyrosine kinases [4–8] or nonreceptor tyrosine kinases (e.g Fyn, Syk [9,10]) and targets them for degradation

We have recently identified a novel adaptor-type protein named Ruk [11] It has been cloned by other groups and named Cin85 or SETA [12,13] Based on sequence homology and domain organization, Ruk L was integrated into a new subfamily of adaptor molecules that includes the protein CD2AP, also named CMS All members of this family contain three SH3 domains at the N-terminus, followed by a proline-rich region, a PEST

towards the C-terminus A variety of signaling molecules, including Grb2, Crk, Sos, Cbl were shown to interact with Cin85 via these protein–protein interaction domains [12,14]

We previously described the existence of three Ruk isoforms named Ruk L, Ruk M and Ruk S, which are products of alternative splicing and differential promoter usage (Fig 1A) These isoforms share a common C-terminal region but are truncated at their N-termini Ruk M possesses only one SH3 domain but all downstream domains are the same as in the Ruk L protein In contrast, Ruk S retains only the C-terminal coiled-coil region

Although the precise role of this Ruk/CD2AP family is still unknown, they are involved in the control of apoptosis and the regulation of cytoskeletal architecture [11,13,15–18]

Correspondence to F Verdier, De´partement d’He´matologie, Institut

Cochin, 27 Rue du Faubourg Saint Jacques, 75014 Paris, France.

Fax: + 33 1 40 51 65 10, Tel.: + 33 1 40 51 65 01,

E-mail: verdier@cochin.inserm.fr

Abbreviations: Ub, ubiquitin; Ruk, regulator of ubiquitous kinase;

PtdIns 3-kinase, phosphoinositol 3-kinase; HA, hemaglutinin

(Received 13 March 2002, revised 14 May 2002,

accepted 30 May 2002)

We found that Ruk L binds to PtdIns-3 kinase via the P85

regulatory subunit of the enzyme and inhibits its catalytic

activity Furthermore, Ruk L overexpression in primary

neurons induces apoptosis, which could be rescued by

coexpression of constitutively activated forms of PtdIns-3

Kinase or its downstream effector PKB/Akt [11] In

agreement with these findings, overexpression of SETA

was shown to trigger apoptosis in astrocytes [13] Specific

associations between SETA and proteins involved in

apoptotic processes, such as AIP1, Alg2, Sb-1 further

confirm its importance in maintaining cellular homeostasis

[13,18] Cin85 has been also implicated in the regulation of

cytoskeletal architecture since it interacts with p130 Cas and

colocalizes with actin cytoskeleton in epithelial cells [18] and

more recently Ruk L has been involved in the regulation of

receptor-mediated endocytosis [19]

Because Ruk L contains PEST sequences that are usually

found in proteins with short half lives [20], and because it

associates with the E3 ubiquitin ligase Cbl, we wondered

whether Ruk proteins are modified by ubiquitination

In this report, we demonstrate that all three Ruk isoforms

are ubiquitinated Co-expression studies in Hek293 cells

allowed us to detect specific association between the E3 Ub

ligase Cbl and all three Ruk isoforms Moreover, binding of

c-Cbl to Ruk M and Ruk S was found to be dependent on

heterodimerization with Ruk L, via its coiled-coil domain

Detailed analysis of the stability of Ruk indicated that

ubiquitination does not trigger its degradation by

proteo-somes

M A T E R I A L S A N D M E T H O D S

Reagents and antibodies

Protease inhibitors N-Ac-Leu-Leu-norLeucinal (ALLN)

and lactacystin were purchased from Sigma and

Calbio-chem, respectively Rabbit polyclonal anti-hemagglutinin

(HA)

4 Ig and anti-Cbl Ig were purchased from Santa Cruz Mouse monoclonal anti-EE

L Stephens (AFRC Babraham Institute, Cambridge) Mouse monoclonal anti-(b-actin) Ig were obtained from Sigma, and mouse anti-(b catenine) Ig from Transduction Laboratories Rabbit polyclonal anti-Ruk antibodies, directed against the C-terminal peptide were produced as described previously [11]

Expression constructs The full-length coding sequences corresponding to all three splicing forms of Ruk (Ruk L, Ruk M and Ruk S) were amplified by PCR using rat cDNAs as templates Amplified cDNA fragments were then cloned into pRc/CMV2 vector (Invitrogen, Life Technologies) in-frame with the N-ter-minal EE-tag epitope (MEFMPME) Generated constructs were verified by restriction enzyme digestion and DNA sequencing pcDNA3/Cbl plasmid was a generous gift from

Y Yarden (The Weizmann Institute of Science, Rehovot, Israel) Mammalian expression vector encoding Ub–HA was a gift from D Bohmann (EMBL, Heidelberg, Germany)

Cell culturing and transient transfection Human embryonic kidney cells (HEK293) were cultured at

37°C and 5% CO2in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Life Technologies, Inc.), 2 mM L-glutamine, 50 UÆmL)1 penicil-lin and 50 lgÆmL)1streptomycin Transient transfections were carried out by using LipofectAMINE according to the manufacturer’s recommendations (Life Technologies, Inc.) Twenty-four hours post-transfection cells were treated with

500 lM cycloheximide, 50 lM ALLN, 50 lM lactacystin,

20 mM NH4Cl, 200 lM chloroquine or vehicle alone for indicated time

Fig 1 Ruk L, Ruk M and Ruk S ubiquitination (A) Schematic representations of the domain organization of the three Ruk proteins SH3, Src homology 3 domain; Pro, proline rich region; PEST, sequences enriched in proline, glutamic acid, serine and threonine; CC, Coiled-Coil domain (B) Hek293 cells were transfected with 1.5 lg of plasmid containing EE-tagged Ruk L cDNA, EE-tagged Ruk M cDNA or EE-tagged Ruk S in the absence or presence of HA-tagged Ub plasmid (1.5 lg) As a control, Hek293 cells were transfected with an empty vector Cell lysates were immunoprecipitated with anti-EE Ig The immunoprecipitates were subjected to immunoblotting with anti-HA antibody (top panel) The positions

of polyubiquitinated Ruk species [(Ub)n-Ruk] are indicated The arrowheads mark the locations of the unmodified forms of Ruk L, M, S The membrane was reprobed with anti-EE antibody (bottom panel).

4°C for 20 min Lysates were cleared by centrifugation for

30 min at 27 000 g and supernatants used for further

experiments Immunoprecipitating antibodies were

incuba-ted with solubilized cell extracts for 1 h at 4°C before the

addition of protein G–Sepharose beads, prewashed in lysis

buffer After a 2-h incubation on the wheel, the beads were

washed three times with lysis buffer and twice with buffer

containing 0.1% Brij 98

from beads by boiling in Laemmli sample buffer and

separated by SDS/PAGE Resolved proteins were

trans-ferred onto a poly(vinylidene difluoride) membrane, which

was incubated for 1 h with blocking solution (5% milk in

Tris/NaCl/0.1%Tween) followed by specific antibody

over-night at 4°C After extensive washing with NaCl/Tris/0.1%

Tween, the membrane was incubated for 1 h with

horse-radish peroxidase-conjugated secondary antibody The

antigen–antibody complexes were detected using enhanced

chemiluminescence (ECL) system (Amersham Pharmacia

Biotech) When immunoblots had to be reprobed, the

membranes were initially stripped and reblocked prior to

incubation with another type of primary antibody

Detection of ubiquitinated proteins

Ub is highly conserved among eukaryotes, as only three of

76 amino acids differ between the human and yeast proteins

Therefore, Ub is not an optimal antigen and anti-Ub Ig

rarely possess good affinity Taking this into account and

that anti-Ruk Ig are not very efficient in

immunoprecipi-tation experiments, we decided to cotransfect Hek293 cells

with plasmids encoding HA-tagged ubiquitin and

EE-tagged versions of Ruk isoforms (EE–Ruk L, EE–Ruk M

and EE–Ruk S) Transiently expressed Ruk isoforms were

immunoprecipitated with anti-EE Ig and resolved by SDS/

PAGE Modification of Ruk by Ub was determined by

immunoblotting with anti-HA Ig

R E S U L T S

All three Ruk isoforms are ubiquitinatedin vivo

Sequence analysis of Ruk L indicated the presence of

multiple PEST motives located between a stretch of

proline-rich sequences and the C-terminal coiled-coil domain The

appearance of PEST motifs in protein sequences is often

associated with reduced protein stability and a short half-life

[20] Taking this into account and the fact that Cin85/SETA

binds the E3 ligase c-Cbl [12,18], we decided to investigate

ubiquitination of Ruk isoforms in vivo In this experiment,

EE-tagged versions of all three isoforms of Ruk were

cotransfected into Hek293 cells together with HA-tagged

Ub Two days after transfection, Ruk isoforms were

immunoprecipitated using anti-EE antibodies, separated

by electrophoresis and analysed by immunoblotting using

anti-HA Ig The results presented in Fig 1B clearly

ance of multiple bands on the anti-HA Western blot, clearly demonstrate that Ruk isoforms are polyubiquitinated

Specific interaction between Ruk isoforms and the E3 ubiquitin ligase Cbl

Specific interaction between c-Cbl and Cin85/SETA, which corresponds to the Ruk L isoform, has been recently reported [12,18] These findings and the results presented above prompted us to investigate whether all Ruk isoforms can interact with Cbl To assess these interactions, EE-tagged versions of Ruk L, M or S were cotransfected into Hek293 cells with c-Cbl or vector alone Immune complexes were precipitated with anti-EE Ig and analysed

by Western blotting using anti-Cbl Ig Figure 2A clearly demonstrates that all three isoforms of Ruk interact with exogenously expressed Cbl Moreover, we also observed coimmunoprecipitation of endogenous Cbl with anti-EE Ig from cells, which were transfected with EE-tagged isoforms

of Ruk Observed interactions were also detected in reciprocal experiments, when anti-Cbl immunoprecipitates were probed in Western blotting with anti-EE Ig Ruk S is detected only on a much longer exposure (Fig 2B) We have also noted that the longest isoform of Ruk (Ruk L) exhibits the strongest association with endogenous and exogenously expressed Cbl The association of Ruk M and Ruk S with c-Cbl was quite unexpected, as these isoforms

do not possess the two N-terminal SH3 domains, found previously to mediate the interaction with c-Cbl [12,18] Heterodimerization of Ruk L with Ruk M and Ruk S All Ruk isoforms share a common C-terminal region which possesses a coiled-coil domain Numerous studies have implicated coiled-coil domains in mediating protein–protein interaction via homodimerizations and heterodimerizations [21] Taking this into account, we speculated that associ-ation of Ruk M and Ruk S with Cbl is not direct, but could

be mediated via heterodimerization with the longest isoform, Ruk L In order to test this hypothesis, we cotransfected nontagged Ruk L together with each EE-tagged Ruk isoforms Following anti-EE Ig immuno-precipitations, immune complexes were subjected to West-ern blot analysis with the C-terminal anti-Ruk Ig, which recognizes all three isoforms As shown in Fig 3A, untagged Ruk L specifically coimmunoprecipitates with all three EE-tagged isoforms The expression of EE-tagged Ruk isoforms in transfected cells was confirmed by immuno-blotting of total cell lysates with anti-EE antibodies (Fig 3B) These data suggest that Ruk isoforms have the potential to form homodimers and heterodimers and that the coiled-coil domain mediates the formation of these complexes Moreover, specific interaction between Cbl and two shorter isoforms of Ruk (Ruk M and Ruk S) was found to be mediated by heterodimerization with Ruk L

Ruk L is ubiquitinated but not degraded

by proteasomes

Ubiquitination, in most cases, targets modified proteins for

degradation by 26S proteasomes In order to determine

whether ubiquitination of Ruk isoforms would induce their

degradation, a panel of proteosome- and lysosome-specific

inhibitors has been used It is well established that

lactacys-tine and the peptide aldehyde ALLN inhibit

proteasome-mediated proteolysis, causing an accumulation of proteins that are usually degraded by this pathway [22] In contrast

to lactacystine, which is a highly specific proteasomal inhibitor [23], ALLN inhibits also nonproteasomal proteases, such as calpains and cathepsins

Initially, we tested the stability of EE-tagged Ruk L transiently overexpressed in Hek293 cells Two days after transfection, cells were treated with various inhibitors or with the vehicle alone Equal amounts of total cell lysates

Fig 3 Heterodimerization of Ruk L with Ruk M and Ruk S Hek293 cells were transfected with 1.5 lg of plasmid containing nontagged version of Ruk L cDNA, and cotransfected with the same quantity of either EE-tagged Ruk L, EE-tagged Ruk M, or EE-tagged Ruk S cDNA As a control, Hek293 cells were transfected with an empty vector (NT) Cell lysates were immunoprecipitated with anti-EE antibody The immunoprecipitates were analysed by Western Blot using anti-Ruk antibody raised against the last 17 C-terminal amino acids and thereby able to recognize all Ruk isoforms (Ruk L, EE-tagged Ruk L, EE-tagged Ruk M and EE-tagged Ruk S) Ruk L and EE-tagged Ruk L can be separated by SDS/PAGE, since their molecular masses differ from approximatly one kDa (A) An aliquot of each cell lysate was immunoblotted with anti-EE Ig to check the expression of EE-Ruk isoforms (B) The blot in the lower panel of (B) was exposed 10 times longer than the upper panel.

Fig 2 Ruk L, Ruk M and Ruk S coprecipitate

with Cbl Hek293 cells were transfected with

1.5 lg of plasmid containing EE-tagged RukL

cDNA, EE-tagged Ruk M cDNA or

EE-tag-ged Ruk S cDNA in the absence or presence of

Cbl plasmid (1.5 lg) As a control, Hek293

cells were transfected with an empty vector

(NT) Cell lysates were split in two and were

immunoprecipitated either with anti-EE Ig (A)

or with anti-Cbl Ig (B) The

immunoprecipi-tates were subjected to immunoblotting with

anti-Cbl Ig (A) or with anti-EE Ig (B).

An aliquot of each cell lysate was

immuno-blotted with anti-EE antibody to check

expression of Ruk isoforms (C).

were separated by SDS/PAGE and immunoblotted with

anti-EE Ig No changes in the level of exogenously expressed

Ruk L was detected, when the activities of proteosomal and

lysosomal proteases were blocked by specific inhibitors

(Fig 4A) We reprobed the membrane with anti-(b-actin) Ig

to confirm equal loading of proteins in each lane (Fig 4A,

lower panel) One can argue that only ubiquitinated form of

Ruk L would be targeted for degradation and if this fraction

is small, it might be difficult to detect the changes in the level

of total Ruk L protein To overcome this problem, we

coexpressed EE-tagged Ruk L with HA-Ub in Hek293

cells Then, cells were treated with proteosomal inhibitor

ALLN or vehicle alone As shown in Fig 4B, no

accumu-lation of ubiquitinated EE-Ruk L is detected in the presence

of ALLN

We then measured the half-life of endogenous Ruk L,

which is expressed at high level in Hek293 cells In most

cases, the half-life of ubiquitinated proteins is short due to

their rapid degradation by the proteasome Time-course

treatment of cells with a protein synthesis inhibitor

cyclo-heximide showed no variations in the level of endogenous

Ruk L, suggesting a long half-life for the protein (Fig 4C)

Furthermore, we examined the effect of selected inhibitors

of protein degradation on the level of endogenous Ruk L

As can be seen in Fig 4D, neither ALLN nor lactacystine

induce any accumulation of endogenous Ruk L in Hek293 cells, even 6 h after treatment These results were also confirmed in human monocytic cell line U937 (data not shown) To verify that proteolytic activities of proteasomes were effectively inhibited by the use of indicated inhibitors,

we checked the expression level of b-catenin, which is degraded via the Ub-proteasome pathway [24,25] Re-probing of the membrane with anti-(b-catenin) Ig clearly demonstrated the accumulation of ubiquitinated forms of b-catenin upon ALLN and lactacystine treatment (Fig 4D, lower panel) No effect on the stability of Ruk L protein was also observed when cells were treated with lysosomal inhibitors: NH4Cl or chloroquine

these experiments clearly demonstrate that neither exogen-ously expressed nor native Ruk L isoform is degraded via proteasome or lysosome pathways

D I S C U S S I O N

Ubiquitination is now recognized as a regulatory protein modification whose functional significance is comparable to that of phosphorylation Degradation of cellular proteins by the ubiquitin system encompasses two successive steps: (a) covalent attachment of ubiquitin molecules to selected proteins; and (b) degradation of ubiquitin-conjugated

Fig 4 Exogenously expressed EE-Ruk L (A,B) or Endogenous Ruk L (C,D) are not degraded by the proteasome (A) Hek293 cells were transfected with 2 lg of plasmid containing EE-tagged Ruk L cDNA After 48h, cells were incubated for the time indicated with various inhibitors or with vehicle alone [Final concentrations: 50 l M for ALLN, 50 l M for Lactacystin (Lacta), 20 m M for NH 4 Cl and 200 l M for chloroquine (Chloro)] Cells were lysed and the quantity of protein measured by Bradford assay 10 lg of total protein from each cell lysate was separated by SDS/PAGE The level of exogenous EE-Ruk L was determined by Western Blot analysis using EE Ig (upper panel) The membrane was reprobed with anti-(b-actin) Ig to confirm equal loading of protein in each lane (bottom panel) (B) Hek293 cells were cotransfected with 1.5 lg of plasmid containing EE-tagged Ruk L cDNA and 1.5 lg of Ub-HA plasmid After 48h, cells were incubated for 3 h with (+) or without (–) ALLN Cell lysates were immunoprecipitated with anti-EE Ig The immunoprecipitates were subjected to immunoblotting with anti-HA Ig The position of polyubiqui-tinated Ruk species [(Ub)n-Ruk] are indicated The arrowheads mark the locations of unmodified EE-Ruk L (C) Hek293 cells were incubated with

500 l M Cycloheximide (CHX) for the times indicated As described in (A), 30 lg of proteins from the total cell lysates was separated by SDS/PAGE electrophoresis, and the expression level of endogenous Ruk L determined by Western blot (WB) using anti-Ruk Ig (upper panel) The membrane was reprobed with anti-(b-actin) Ig to confirm equal loading of protein in each lane (lower panel) (D) Hek293 cells were incubated for the time indicated with various inhibitors or with vehicle alone as described for panel A Cells were lysed and the quantity of proteins measured by Bradford assay 30 lg of proteins from the total cell lysates was separated by SDS/PAGE electrophoresis The expression level of endogenous Ruk L was determined by Western blot analysis using anti-Ruk Ig (upper panel) The membrane was reprobed with anti-(b-catenin) Ig (bottom panel) The position of polyubiquitinated b-catenin species [(Ub)n-b-catenin] are indicated The arrowheads mark the locations of unmodified b-catenin.

proteins Many ubiquitinated proteins are targeted for

degradation by 26S proteasomes, but some undergo

endocytosis, leading to proteolysis in the lysosome New

findings show that ubiquitination is not always associated

with the degradation of modified proteins, but could be also

involved in regulation of enzymatic activities and

intranu-clear trafficking of tagged proteins [26]

The data presented in this study clearly demonstrate that

a recently identified adaptor-type protein Ruk L, also

known as Cin85 or SETA, is ubiquitinated in vivo

Furthermore, we showed that shorter splicing variants of

Ruk, termed Ruk M and Ruk S, are also modified by

covalent attachment of ubiquitin Ubiquitination of all three

isoforms of Ruk indicates that ubiquitin conjugation occurs

at the C-terminal region, which is common between them It

is well established that ubiquitin is conjugated to target

proteins through lysine residues Sequence analysis of Ruk

isoforms showed that their common C-terminal region

contains numerous lysine residues, which could be potential

sites for ubiquitination The identification and

characteri-zation of Ruk ubiquitination sites is currently in progress

Recently, specific association between Cbl and Cin85/

SETA was demonstrated [12,18] This interaction was found

to be mediated by the first two SH3 domains of Cin85/

SETA and the proline-rich region of Cbl In addition to

that, constitutive binding between both proteins was further

induced by stimulation of cells with EGF and found to be

dependent on tyrosine phosphorylation of the C-terminal

region of Cbl ([12] and our data, not shown) The same

mode of interaction has been recently reported between Cbl

and CMS/CD2AP, which requires the same domains and is

also regulated by tyrosine phosphorylation of Cbl [27] It is

believed that tyrosine phosphorylation at the C-terminus of

Cbl induces a conformational change of the protein from a

closed to an open conformation, thereby unmasking

putative SH3-domain motifs

In agreement with these findings, we show, in vivo, an

interaction between Cbl and the longest isoform of Ruk,

Ruk L Furthermore, specific association of Cbl with

Ruk M and Ruk S was also demonstrated As neither

Ruk M nor Ruk S possess the first two SH3 domains,

which are involved in complex formation with Cbl, the

mechanism of these interactions has been investigated We

found that the C-terminal region, which contains a

coiled-coil domain common to all Ruk isoforms, is responsible for

their heterodimerization These results suggest that binding

between Cbl/Ruk M and Cbl/Ruk S are indirect and

require heterodimerization with Ruk L

As all isoforms are in complex with Cbl and are

ubiquitinated, Cbl is a potential E3 ligase responsible for

ubiquitination of Ruk isoforms This hypothesis is currently

under investigation

The discovery of Ruk isoform ubiquitination in cells

prompted us to investigate the importance of this

modifi-cation, especially in regulating its stability Using

proteo-somal and lysosomal inhibitors, we found that

ubiquitination of exogenously expressed and native Ruk L

does not induce its degradation via these proteolytic

pathways If ubiquitination of Ruk L is not a signal for

its degradation, what is the role of this post-translational

modification?

Proteolysis-independent regulation by ubiquitination has

recently been reported in several systems Ubiquitination of

a number of cell surface receptors in response to ligand binding serves as an internalization signal [28] Moreover, ubiquitination-dependent processing of precursor proteins [29] and the regulation of multienzyme complex formation have also been described [30] An interesting paper by Fang

et al demonstrates that like Ruk L, the P85 regulatory subunit of PtdIns-3 kinase is ubiquitinated, but not degraded by the proteasome pathway [31,32] In addition, Cbl was shown to be the E3 ligase responsible for P85 ubiquitination and to regulate the recruitment of PtdIns-3 kinase to CD28and T cell antigen receptor complexes, thereby inhibiting PtdIns-3 kinase activation

Two contradictory mechanisms have been proposed for Cbl binding to the P85 subunit: one involves the proline-rich domain of Cbl and the SH3 domain of P85 and while the other implicates a phosphorylated C-terminal tyrosine (Y731) of Cbl and an undefined domain of P85 [32,33]

We previously reported specific association between Ruk L and the P85 regulatory subunit of PtdIns-3 kinase, which is mediated via proline-rich sequences and the SH3 domain, respectively [11] This interaction is not inducible by growth factor stimulation and has an inhibitory effect on the activity of PtdIns-3 kinase The mechanism by which PtdIns-3 kinase could be released from the inhibitory complex with Ruk L is still unknown Ruk L polyubiquiti-nation could induce conformational changes in the molecule which may modify its binding specificity towards the p85 subunit of the PtdIns-3 Kinase We are currently investi-gating whether ubiquitination of Ruk L and P85 could affect the association between them and the activity of PtdIns-3 kinase The use of in vitro binding and ubiquiti-nation assays will allow us to better understand the mechanism of the interaction between Ruk L, E3 ligase Cbl and PtdIns-3 kinase

A C K N O W L E D G E M E N T S

Fre´de´rique Verdier is supported by an EMBO (European Molecular Biology Organization) Fellowship We are grateful to Mark Griffin for expert technical assistance and to H Rebholz and T Fenton for critical reading of the manuscript.

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