The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Accession:
Functional site class:
CIN85 and CD2AP SH3 domain binding motif
Functional site description:
CIN85 is an adaptor protein with many interaction partners most notably the E3 ligase Cbl involved in membrane trafficking regulation. CIN85 is particularly involved in endocytosis and cytoskeletal rearrangement. CIN85 participates in the regulation of receptor tyrosine kinase (RTK) signalling and affects kidney function, T- and B-cell receptor signalling and apoptosis in neuronal cells. CD2AP is a close homologue of CIN85 with both proteins containing three SH3 domains. The three SH3 domains of CIN85 and the first two of CD2AP recognize their binding partners through a non-canonical proline-rich PxpxPR motif, forming complexes connecting interaction partners to form functional assemblies: some of these have been identified as phase-separated liquid condensates. CIN85 and CD2AP represent a direct link between plasma membrane receptors, like the BCR complex and CD2, and the actin-cytoskeleton network. PxpxPR is also present in pathogen proteins which hijack CIN85/CD2AP.
ELM Description:
PxpxPR is an atypical SH3 domain-binding motif. Alanine can replace proline in the 3rd position without significant loss of affinity (Rouka,2015) and is observed in several known binding partners. Although the SH3 domains of CIN85 and CD2AP might also accommodate Px[VI]xPR motifs (Rouka,2015) as mentioned in conjunction with PAK1 binding (Kurakin,2003; Jozic,2005), these interactions are low in affinity and their functional relevance is questionable (Ile at the +3 position may be specific for interaction with a homologous SH3 domain in ARHGEF7). In CIN85, all three SH3 domains bind the Px[PA]xPR motif (Kurakin,2003). However, in CD2AP only the first two SH3s bind this motif, while the third belongs to another SH3 subfamily, was proposed to bind another, extended motif pattern based on mutational peptide array analysis (Rouka,2015) and only showed binding to Px[PA]xPR motif-harbouring peptides with an embedded PxxP motif in a peptide array study (Rouka,2015).

For c-Cbl and Cbl-b, binding in class I and the inverted class II orientation is simultaneously possible. This is due to the pseudo-symmetric sequence formed by an additional arginine residue close to the amino-terminus of the motif (Jozic,2005; 2BZ8; 2AK5). However, it was shown that this extension of the recognition sequence Px[PA]xPR to RxPxxxPR is necessary for enabling the class I interaction. Most proteins do not have this Rx extension and are not capable to interact with the hydrophilic pocket in the SH3 domain, resulting in class II orientated interactions only. Further, the K907 in Cbl-b stabilizes the type I interaction with SH3 domains. Results from NMR, ITC, and crystallography show that the interaction mechanism also depends on the adaptor protein. Steric differences associated with the n-Src loop of CIN85 and CD2AP seem to have an impact on the formation of a dimeric or trimeric complex, with trimeric complex formation with Cbl-b only being feasible for CIN85 (Ceregido,2013).
Pattern: P.[AP].PR
Pattern Probability: 0.0000956
Present in taxons: Alphavirus Apicomplexa Chikungunya virus Human herpesvirus 5 Theileria annulata Toxoplasma gondii Vertebrata Viruses
Interaction Domain:
SH3_9 (PF14604) Variant SH3 domain (Stochiometry: 1 : 1)
o See 60 Instances for LIG_SH3_CIN85_PxpxPR_1
o Abstract
Most of the SH3-binding partner motifs match the canonical PxxP minimal consensus sequence which is accommodated on the SH3 surface in two xP dipeptide-binding pockets (LIG_SH3_1; LIG_SH3_2; LIG_SH3_3). As is not unusual for linear motif interactions, SH3-interacting motifs can bind to the domain interaction surface in two opposite orientations, termed class I or class II. The non-canonical binding motif PxpxPR discussed here does not contain the PxxP consensus sequence, thus is unlikely to adopt the typical polyproline type II (PPII) helical conformation and mainly binds in the Class II orientation (Saksela,2012). Being a proline-rich motif, it is very likely to be found in flexible natively disordered polypeptide regions rather than within folded domains, thus being accessible as a binding motif.

This non-canonical SH3 binding motif is used in multiple cellular processes of vertebrate organisms. The adaptor protein CIN85 (Cbl-interacting protein of 85 kDa, also known as SH3KBP1; SH3K1_HUMAN) and its close homologue CD2AP (CD2-associated protein; CD2AP_HUMAN) contain three SH3 domains that bind to proteins containing the proline-rich PxpxPR motif (Kurakin,2003; Rouka,2015) and are the main PxpxPR binding partners. Several studies have shown that a combination of the SH3 domains cooperatively bind to targets more strongly than a single SH3 interaction (Kirsch,2001; Konishi,2006; Sato,2007). The two rather large adaptor proteins CIN85 and CD2AP themselves contain a proline-rich region that is bound by SH3 domains of several kinases and adaptor proteins plus a coiled-coil region that mediates homodimerization (Dikic,2002). CIN85 recruits a variety of proteins, most of them associated with membranes and/or the cytoskeleton (Havrylov,2010). Since CIN85 is able to dimerize and use its SH3 domains simultaneously it can bind multiple molecules of the same protein, thus serving as an adaptor that promotes clustering in different process-dependent contexts such as the control of RTK signalling, actin reorganization, T cell functions, kidney architecture and apoptotic signalling (Kowanetz,2003). The interaction of the PxpxPR containing Cbl proteins with the CIN85 SH3 domains plays multiple roles in RTK down-regulation, endocytosis and degradation (Soubeyran,2002; Kowanetz,2003; Jozic,2005; Havrylov,2010). Furthermore, the c-Cbl CIN85 interaction is involved in vesicle mediated endocytosis that is initiated through the ubiquitination of cell surface receptors by c-Cbl (Kowanetz,2004; Havrylov,2010). PxpxPR motif-harbouring partners such as ARAP1, SEPT9 and SHKBP1 are reported to negatively regulate epidermal growth factor receptor (EGFR) endocytosis and degradation by preventing the association of Cbl with CIN85 (Feng,2011; Diesenberg,2015; Li,2018). CD2AP has similar roles as CIN85 in the regulation of RTKs. They are also known to regulate actin cytoskeleton dynamics through direct binding and regulation of the actin capping protein (LIG_ActinCP_CPI_1; Hutchings,2003; Huber,2018). The PxpxPR-mediated interaction of CD2AP with CD2 is crucial for CD2 clustering. This process mediates T-cell polarization as well as recruitment of CD2 during the formation of an immunological synapse (Ceregido,2013). It is known that CD2AP and CIN85 are themselves also able to interact and can together promote cross-linking of actin filaments into bundles indicating a cytoskeleton modulating activity (Havrylov,2010). Both, CD2AP and CIN85 associate with the cytosolic domain of the T-cell surface protein CD2 upon T-cell activation (Ceregido,2013).

CIN85 and CD2AP are large, modular, multi-domain proteins with several copies of SH3 domains and proline-rich motifs. Due to their multivalent interactions and ability to homo-oligomerize, they are perfect candidates for driving liquid-liquid phase separation (LLPS). Indeed, it has been recently demonstrated that in B cells, CIN85 trimerizes through its C-terminal coiled-coil domain and the resulting trimeric CIN85 molecules associate with multiple copies of PxpxPR motifs within several SLP-65 molecules (Src homology (SH) 2 domain-containing leukocyte protein of 65 kDa, also called B-cell linker protein, BLNK; Q8WV28) that recruite additional CIN85 trimers. The resulting extended network of molecular scaffolds (Kuhn,2016) shows the hallmarks of phase-separated liquid condensates (Wong,2020) and is crucial for the efficient initiation of intracellular signalling upon BCR stimulation.

PAK1 contains a PxIxPR motif that is normally recognized by the ARHGEF7 SH3 domain but can be also bound by the second SH3 domain of CIN85, indicating that isoleucine might also be tolerated in the third position of the motif (Jozic,2005). Motifs with valine in the third position were also reported to be recognized by CIN85 (Haglund,2005). However, the binding to CIN85 is low affinity and it is unsure if this is the relevant binding motif (Saksela,2012) and needs to be confirmed by examining further instances.

Strikingly, in comparison to other SH3 domain binding peptides, binding in class I and class II orientation is simultaneously possible (Jozic,2005; 2BZ8; 2AK5) due to the pseudo-symmetric sequence in c-Cbl (P22681) and Cbl-b (Q13191) (RxPxPxPR). However, binding orientation depends on steric properties of the adaptor protein (Ceregido,2013). The binding affinity is in the low micromolar range for individual SH3 domain-PxpxPR interactions, comparable to affinities of other SH3 domains binding to classical polyproline peptides (Kowanetz,2003). Binding stoichiometry can be 2:1 (two SH3 domains held together by a single pseudo-symmetrical peptide sequence of Cbl (2BZ8; Jozic,2005)), or 1:1 depending on steric and sequential properties.

As shown more recently, the functional SH3 domain-binding motif (PxpxPR) is also involved in hijacking signalling pathways with important functions for pathogen infections, as in Theileria annulata-infected cells (Huber,2018) or in infection by Toxoplasma gondii (Guerin,2017). Furthermore, it can be associated with viral infected cells as shown for the chikungunya virus (Agback,2019), human cytomegalovirus (Rak,2018), Hepatitis C virus (Igloi,2015) and Herpes simplex virus 1 (Liang,2005). The protein TA20980 (Q4UGW0) secreted by Theileria annulata interacts specifically with bovine CD2AP (F1MM14) via its functional SH3 domain-binding motif (Huber,2018). Both, CD2AP and CIN85 are engaged in the invasion mechanism used by the apicomplexan parasite Toxoplasma gondii to force the parasite into the host cell (Guerin,2017). Since CD2AP is known to connect cortical cytoskeleton to cytosolic domains of transmembrane proteins, the recruitment of CD2AP to the RON complex of Toxoplasma gondii allows for physically linking it to the host cytoskeleton (Guerin,2017). In chikungunya virus infection, CD2AP has an impact on the initiation of viral replication (Agback,2019). The motif PxxxPR is found in the intrinsically disordered, hypervariable domain of the NSP3 protein of a subgroup of alphaviruses and binds CD2AP (Mutso,2018), an interaction that has been studied mainly in the chikungunya NSP3 protein (A3RMR8; Agback,2019). Human cytomegalovirus encodes for the pUL135 protein (Q64ET4) which contains five putative PxxxPR SH3 domain-binding motifs to interact with CIN85 (Q96B97) and a KxxPxxP motif to bind Abi-1 (Q8IZP0) to take control over EGFR regulation and trafficking in the infected cell. For reactivation of human cytomegalovirus from latency, pUL135 is required to interact with both CIN85 and Abi-1 (Rak,2018). Hepatitis C virus (HCV) NS5A protein impairs epidermal growth factor (EGF) receptor (EGFR) endocytosis and degradation through its interaction with CD2AP (Igloi,2015), while Herpes simplex virus 1 infected cell protein 0 (ICP0) binds CIN85 and Cbl to mediate the clearance of EGF receptor from cell surfaces by promoting its degradation (Liang,2005).

o 20 selected references:

o 17 GO-Terms:

o 60 Instances for LIG_SH3_CIN85_PxpxPR_1
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
P22681 CBL
CBL_HUMAN
824 829 SQVPERPPKPFPRRINSERK TP 16 Homo sapiens (Human)
4 
Q13191 CBLB
CBLB_HUMAN
906 911 SQAPARPPKPRPRRTAPEIH TP 22 Homo sapiens (Human)
4 
P06729 CD2
CD2_HUMAN
327 332 VHQQKGPPLPRPRVQPKPPH TP 14 Homo sapiens (Human)
4 
Q8TB24 RIN3
RIN3_HUMAN
457 462 PRTAKQPPVPPPRKKRISRQ TP 12 Homo sapiens (Human)
2 
Q8TB24 RIN3
RIN3_HUMAN
383 388 LPAKKNLPTAPPRRRVSERV TP 12 Homo sapiens (Human)
2 
S8ERS3 RON5
S8ERS3_TOXGM
94 99 RQHGSGPPRPAPRRAAAVAD TP 6 Toxoplasma gondii ME49
5 
Q9ULH1 ASAP1
ASAP1_HUMAN
1055 1060 TPTLPETPVPLPRKINTGKN TP 2 Homo sapiens (Human)
3 
Q9UHD8 SEPTIN9
SEPT9_HUMAN
143 148 EVLGHKTPEPAPRRTEITIV TP 4 Homo sapiens (Human)
1 
Q9QZA2 Pdcd6ip
PDC6I_RAT
744 749 AGGHATAPTPAPRTMPPAKP TP 6 Rattus norvegicus (Norway rat)
1 
Q9QWY8 Asap1
ASAP1_MOUSE
1073 1078 TPTLPETPVPLPRKINTGKN TP 4 Mus musculus (House mouse)
1 
Q9JM90 Stap1
STAP1_MOUSE
7 12 MMAKKPPKPAPRRIFQERLK TP 2 Mus musculus (House mouse)
3 
Q9JKY5 Hip1r
HIP1R_MOUSE
1026 1031 SEEEPSRPSPAPRSGATKKP TP 2 Mus musculus (House mouse)
3 
Q99IB8 Genome polyprotein
POLG_HCVJF
2323 2328 LPPPKKAPTPPPRRRRTVGL TP 3 Hepatitis C virus JFH-1
2 
Q96J02 ITCH
ITCH_HUMAN
260 265 PPRPSRPPPPTPRRPASVNG TP 1 Homo sapiens (Human)
1 
Q96EY5 MVB12A
MB12A_HUMAN
155 160 KKAKAPRPVPKPRGLSRDMQ TP 4 Homo sapiens (Human)
2 
Q92835 INPP5D
SHIP1_HUMAN
1135 1140 EINQQTPPTPTPRPPLPVKS TP 2 Homo sapiens (Human)
3 
Q92835 INPP5D
SHIP1_HUMAN
1029 1034 YGSLSSFPKPAPRKDQESPK TP 2 Homo sapiens (Human)
3 
Q8WWN8 ARAP3
ARAP3_HUMAN
129 134 GPGVSRSPEPSPRPPPLPTS FP 2 Homo sapiens (Human)
3 
Q8WWN8 ARAP3
ARAP3_HUMAN
94 99 PQAQPPKPVPKPRTVFGGLS TP 2 Homo sapiens (Human)
2 
Q8WV28 BLNK
BLNK_HUMAN
307 312 QKQIHQKPIPLPRFTEGGNP TP 6 Homo sapiens (Human)
3 
Q8WV28 BLNK
BLNK_HUMAN
242 247 KSPPPAAPSPLPRAGKKPTT TP 8 Homo sapiens (Human)
3 
Q8WUM4 PDCD6IP
PDC6I_HUMAN
740 745 GGHAPTPPTPAPRTMPPTKP TP 12 Homo sapiens (Human)
2 
Q8JUX6 Polyprotein P1234
POLN_CHIKS
1731 1736 DWVMSTVPVAPPRRRRGRNL TP 3 Chikungunya virus strain S27-African prototype
2 
Q8CHC4 Synj1
SYNJ1_MOUSE
1282 1287 QPNLETPPQPPPRSRSSQSL TP 3 Mus musculus (House mouse)
3 
Q6TLK4 Arhgap27
RHG27_RAT
448 453 QWELPQVPVPAPRSGRKSSQ TP 4 Rattus norvegicus (Norway rat)
1 
Q6P7W2 Shkbp1
SHKB1_MOUSE
678 683 APELRWPPTPAPRPSTSLGN TP 2 Mus musculus (House mouse)
3 
Q6P7W2 Shkbp1
SHKB1_MOUSE
618 623 ASSRGSFPSPSPRTSLTSLH TP 2 Mus musculus (House mouse)
3 
Q6ISU1 PTCRA
PTCRA_HUMAN
211 216 GREATSSPRPQPRDRRWGDT TP 6 Homo sapiens (Human)
2 
Q64ET4 UL135
Q64ET4_HCMV
277 282 SKVGLSCPCPRPRTPTEPTT U 4 Human herpesvirus 5 (Human cytomegalovirus)
3 
Q64ET4 UL135
Q64ET4_HCMV
209 214 SSHRPPTPIPAPRKNLSTPP U 4 Human herpesvirus 5 (Human cytomegalovirus)
3 
Q4UGW0 TA20980
Q4UGW0_THEAN
220 225 VTIEKPSPTPKPRTKPGTME U 3 Theileria annulata
1 
Q4UGW0 TA20980
Q4UGW0_THEAN
204 209 DTKPKPSPIPKPRTKPVTIE U 4 Theileria annulata
2 
Q4UGW0 TA20980
Q4UGW0_THEAN
188 193 PEGVPQRPTPKPRTKPDTKP U 4 Theileria annulata
2 
P98082 DAB2
DAB2_HUMAN
715 720 LNKINEPPKPAPRQVSLPVT TP 6 Homo sapiens (Human)
3 
P98082 DAB2
DAB2_HUMAN
620 625 SSLLVTPPQPPPRAGPPKDI FP 3 Homo sapiens (Human)
3 
P97318 Dab1
DAB1_MOUSE
516 521 TPSTNSPPTPAPRQSSPSKS TP 6 Mus musculus (House mouse)
3 
P87515 Polyprotein P1234
POLN_BFV
1737 1742 EVHQAPPTPVPPPRPKRAAK TP 2 Barmah Forest virus
2 
P87515 Polyprotein P1234
POLN_BFV
1703 1708 PAPRTIFRPVPAPRAPVLRT TP 2 Barmah Forest virus
2 
P87515 Polyprotein P1234
POLN_BFV
1693 1698 SLPVGDTRPIPAPRTIFRPV TP 2 Barmah Forest virus
2 
P78314 SH3BP2
3BP2_HUMAN
208 213 PPAYPPPPVPTPRKPAFSDM TP 5 Homo sapiens (Human)
1 
P27986 PIK3R1
P85A_HUMAN
85 90 GRKKISPPTPKPRPPRPLPV TP 3 Homo sapiens (Human)
1 
P27282 Polyprotein P1234
POLN_EEVVT
1799 1804 RTVFRNPPHPAPRTRTPSLA TP 3 Venezuelan equine encephalitis virus (strain Trinidad donkey)
4 
P27282 Polyprotein P1234
POLN_EEVVT
1787 1792 SMEFLARPVPAPRTVFRNPP TP 3 Venezuelan equine encephalitis virus (strain Trinidad donkey)
4 
P26651 ZFP36
TTP_HUMAN
309 314 GVFAPPQPVAAPRRLPIFNR TP 6 Homo sapiens (Human)
3 
P0C5J9 Shkbp1
SHKB1_RAT
678 683 APELRGPPTPAPRPSTSLGN TP 4 Rattus norvegicus (Norway rat)
2 
P0C5J9 Shkbp1
SHKB1_RAT
618 623 TSSRASFPSPSPRTSLTSLH TP 4 Rattus norvegicus (Norway rat)
2 
P08920 Cd2
CD2_MOUSE
320 325 IHQQKGPPLPRPRVQPKPPC TP 4 Mus musculus (House mouse)
1 
P08411 Non-structural polyprotein
POLN_SFV
1771 1776 PVPAPRKPTPAPRTAFRNKL TP 4 Semliki forest virus
4 
P08411 Non-structural polyprotein
POLN_SFV
1764 1769 ASRAAERPVPAPRKPTPAPR TP 4 Semliki forest virus
4 
P08393 ICP0
ICP0_HHV11
414 419 PCVRAPPPGPGPRAPAPGAE TP 5 Herpes simplex virus (type 1 / strain 17)
3 
P08393 ICP0
ICP0_HHV11
253 258 PPAPRRTPRAPPRRGAAAPP TP 5 Herpes simplex virus (type 1 / strain 17)
3 
O94850 DDN
DEND_HUMAN
143 148 RRSPPGRPRPEPRNAPRVAQ TP 4 Homo sapiens (Human)
1 
O75553 DAB1
DAB1_HUMAN
516 521 TPSTNSPPTPAPRQSSPSKS TP 2 Homo sapiens (Human)
3 
O55207-2 Synj2
SYNJ2_RAT
1244 1249 PITAPIPPVPKPRTFQPGRG TP 2 Rattus norvegicus (Norway rat)
3 
B6KV60 RON2
RON2_TOXGM
59 64 DATPGLRPQPSPRTFRPTGY TP 6 Toxoplasma gondii ME49
5 
B6KJ32 RON4
RON4_TOXGO
260 265 GEGESQPPTAAPRTSRSVDT TP 1 Toxoplasma gondii
1 
B6KJ32 RON4
RON4_TOXGO
131 136 GEGERQPPTAAPRTSRSVDT TP 1 Toxoplasma gondii
1 
B6KJ32 RON4
RON4_TOXGO
115 120 VPQLGTPPRPAPRRSKGEGE TP 1 Toxoplasma gondii
1 
A0A1B0SZV8 cd2
A0A1B0SZV8_ORENI
352 357 GGEEQPPPLPQPRKKTPKAP TP 2 Oreochromis niloticus (Nile tilapia)
2 
A0A1B0SZV8 cd2
A0A1B0SZV8_ORENI
328 333 PEPANGQPQPSPRRATQASA TP 2 Oreochromis niloticus (Nile tilapia)
2 
Please cite: ELM — the eukaryotic linear motif resource in 2020. (PMID:31680160)

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