Post-translational Modifications Regulate Ral GTPases

 

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RalA and RalB GTPases regulate cell motility, morphology, signaling, vesicular trafficking, and endo/exocytosis. The regulation of these functions is critical for the development and spread of cancer1-4, implicating Ral in oncogenesis and metastasis. Both isoforms are integral for Ras-mediated tumorigenesis, metastasis, and invasion2-6. Despite sharing 82% amino acid sequence identity, effectors, and structural/biochemical properties4, RalA and RalB have their own unique functions in oncogenesis due to distinct subcellular localization and differential effector interactions2,4,7,8. Ral localization, binding partners, and function are regulated by post-translational modifications (PTMs)2,4,9-11. Ral PTMs are found within the Switch 1 and C-terminal hypervariable regions and include geranylgeranylation, carboxymethylation, palmitoylation, phosphorylation, and ubiquitination3,4 (Tables 1 and 2). 

 

Geranylgeranylation

Geranylgeranylation (GG) is necessary for proper membrane localization and functionality of Ral12,13. Geranylgeranylated RalA and RalB are both similarly localized to the plasma membrane13; however, others have reported differential localization11. Both isoforms terminate in a CAAX motif (C=cysteine, A=aliphatic amino acid, X=terminal amino acid), the signal motif for GG4. GG is followed by carboxymethylation of the modified now terminal cysteine residue following enzymatic cleavage of the AAX residues4,14. A subset of Ral proteins express the alternative CCAX motif, which signals modification by palmitoylation instead of carboxymethylation on the second cysteine4,15. Formation of two differentially modified Ral proteins raises therapeutic dilemmas because CAAX processing enzymes are druggable targets16,17. Proteins with the CCAX motif are likely immune to such treatments.


Inhibition of GG by either mutation of the cysteine residue or pharmacological inhibition of geranylgeranyltransferase (a.k.a. GGTase I) disrupts Ral association with the membrane and functionality13. GGTase I inhibitors target both isoforms; however, differential effects follow the inhibition13. GGTase I inhibitors prevent plasma membrane localization of either Ral isoform; instead the Ral GTPases are found throughout the cytoplasm and perinuclear region13. Additionally, a GGTase I inhibitor significantly decreases proliferation of various cancer cell lines in vitro18,19, an effect replicated by genetic ablation of RalA18. Similar GGTase I inhibitor-mediated anti-tumorigenic effects are observed in vivo19.

 

Table 1. Ral Isoforms, PTMs, and Enzymes

Ral Isoform PTM Enzymes Modified Ral aa Ref
RalA Phosphorylation / Dephosphorylation AKA / PP2A Aβ Ser194 21
RalA Phosphorylation / Dephosphorylation
Unknown / PP2A Aβ Ser183 20
RalB
Phosphorylation / Dephosphorylation
PKC / Unknown Ser198 24
RalB
Phosphorylation / Dephosphorylation
PKC alpha / Unknown Ser192 9
RalB
Phosphorylation / Deubiquitination
Unknown / USP33 Lys47 26

aa, amino acid; Ser, serine; Lys, Lysine

 

Table 2. Ral Switch 1 and Hypervariable Regions

 RalA 

 EDYEPTKADSY

 ARKMEDSKEKNGKKKRKSLAKRIRERCCI

RalB

 EDYEPTKADSY

 TKKMSENKDKNGKKSSKN-KKSFKERCCL

Legend: Bold residues are Switch 1. Others are Hypevariable Region. Red indicates sequence overlap. Phosphorylation sites are green. Ubiquitination site is purple.

 

Phosphorylation

The Ral isoforms undergo phosphorylation on different serine residues mediated by different kinases in vitro.  The C-terminal RalA S18320 and S19421 residues are phosphorylated, which activates RalA10,21. Both are specifically dephosphorylated by PP2A Ab, leading to decreased RalA activity20. Loss of PP2A Aβ expression increases phosphorylation of S194 and S183, increasing RalA activation and inducing RalA-mediated cell transformation20. The kinase responsible for phosphorylating S183 is unknown22, while S194 is phosphorylated by Aurora A kinase (AAK)21. Besides increasing activity, AAK-mediated phosphorylation of S194 causes trafficking of active RalA and the effector RalBP1 to internal membranes, followed by increases in RalA/RalBP1-mediated downstream signaling10. For example, phosphorylated RalA and RalBP1 re-localize to the mitochondria itself or mitochondrial vesicles, initiating a signaling cascade that results in mitotic mitochondria fission23. Upon phosphorylation, RalA preferentially binds RalBP1 rather than exocyst subunits Sec5 or Exo8410. In addition, phosphorylation of S194 regulates the in vivo and in vitro oncogenic functions of RalA10,21. RalA is also phosphorylated by PKA22 on yet to be described residues. AKA inhibitors significantly decrease proliferation of cancer cells in vitro18,19, reduce in vivo tumorigenesis19,  and prevent RalA activation in cancer cells19


RalB is phosphorylated by PKC on S19824 and PKC alpha on S1929. Phosphorylation of S198 stimulates activation of RalB9, causes relocalization from plasma membrane to the perinuclear region24, including endocytic vesicles9, and regulates RalB oncogenic functions such as anchorage-independent growth, cell motility, and actin cytoskeleton dynamics in vitro24. In vivo, RalB S198 phosphorylation is necessary for tumor growth and metastasis24. Additionally, S198 phosphorylation alters RalB’s effector interactions. Phospho-mimetic RalB is bound to RalBP1 preferentially at the endomembranes while phospho-deficient RalB is bound to Sec5 at the plasma membrane. The phosphorylation state of RalB regulates vesicle trafficking to and fusion with the plasma membrane and the subsequent release of vesicle cargo proteins9.

 

Ubiquitination

Ubiquitination identifies proteins destined for proteasome-mediated degradation as well as regulating a protein’s activity, localization, or binding partner(s). The Ral isoforms are mainly monoubiquitinated in a nondegradative manner independent of activation state25. However, RalA, but not RalB, is modified by nondegradative ubiquitination under anchorage-independent conditions. While both Ral isoforms can be found at the plasma membrane and endomembranes, constitutively ubiquitinated Ral constructs induce robust plasma membrane enrichment of RalA, but not RalB, which is localized as distinct puncta intracellularly. RalA ubiquitination triggers lipid raft enrichment at the plasma membrane. RalA is deubiquitinated in the raft microdomains which is necessary for endocytosis of the rafts25. In a cyclical manner, endocytosis of lipid rafts caused an increase in RalA ubiquitination while blockade of endocytosis triggered a decrease. Several lysine residues are viable sites for  Ral ubiquitination25. Ubiquitination of the Lys47 RalB residue determines effector binding and subsequent functionality. When ubiquitinated, RalB binds to Sec5 rather than Exo84; however, upon deubiquitination, RalB binds to Exo84 preferentially26. Binding to the former effector mediates innate immune response signaling while binding to the latter mediates autophagocytosis26

 

Conclusion

Ral GTPases undergo a multitude of PTMs that regulate their activity, subcellular localization, effector binding, and ultimately, function. Given the involvement of Ral proteins in Ras-mediated oncogenesis, there is a clear impetus to develop therapeutics that target Ral activation27. Besides directly targeting Ral binding to downstream effectors27, another therapeutic avenue is modulation of Ral activity by increasing or decreasing certain PTMs. To study this untapped therapeutic potential, Cytoskeleton offers Ral activation assays and monoclonal antibodies that specifically target such PTMs as acetylation, phosphorylation, and ubiquitination.

 


 

Related Products & Resources

NEW! Anti-Acetyl Lysine Mouse Monoclonal Antibody (Cat. # AAC01)

NEW! Anti-Phosphotyrosine Mouse Monoclonal Antibody (Cat. # APY03)

NEW! Anti-Ubiquitin Mouse Monoclonal Antibody (Cat. # AUB01)

RhoA G-LISA Activation Assay (Cat. # BK129)

RhoA Pull-down Activation Assay (Cat. # BK040)

 

Activation Assay Video

Cytoskeleton's Small G-protein Activation Assays come in a traditional pull-down bead format or our advanced ELISA based G-LISA format.  Learn which Activation Assay format is right for you by watching this video.

 

 


 

References

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