Biomedical subjects

Nin-Nin Chuang

Publications and source records attributed to Nin-Nin Chuang.

9 recordsLinked to original sources

PubMed2006

Shift syndecan-2 from RACK1 to caveolin-2 upon transformation with oncogenic ras.

Syndecan-2 was found to detach from RACK1 and associate with caveolin-2 and Ras in cells transformed with oncogenic ras. Most of syndecan-2 from transformed cells was revealed with negligible phosphorylations at tyrosine residues. We experimented with HeLa cells transfected with plasmids encoding syndecan-2 and its mutants (syndecan-2(Y180F), syndecan-2(Y192F), and syndecan-2(Y180,192F)) to provide evidences that PY180 of syndecan-2 is a binding site for RACK1 and is deprived in cells transfected with oncogenic ras. However, in HeLa cells transfected with syndecan-2(Y180F), RACK1 was found to sustain its reactions with syndecan-2 independent of phosphorylation. The finding of syndecan-2 reactive with caveolin-2/Ras suggests the molecular complex most likely to obstruct RACK1 for functional attachment at syndecan-2, as revealed in cells transfected with oncogenic ras. We provided evidences to reinforce the view that molecular rearrangements upon transformation are specific and interesting.

Animals↗

PubMed2005

Trap RACK1 with Ras to mobilize Src signaling at syndecan-2/p120-GAP upon transformation with oncogenic ras.

HiTrap-syndecan-2/p120-GAP and HiTrap-syndecan-2/RACK1 affinity columns were applied to reveal that Src tyrosine kinase was highly expressed in BALB/3T3 cells transfected with plasmids pcDNA3.1-[S-ras(Q(61)K)] of shrimp Penaeus japonicus. Both columns were effective to isolate Src tyrosine kinase. The selective molecular affinity for Src was found to be stronger with HiTrap-syndecan-2/RACK1, as revealed with competitive RACK1 to dislodge Src from HiTrap-syndecan-2/p120-GAP. We thus challenged the syndecan-2/p120-GAP and syndecan-2/RACK1 with GTP-K(B)-Ras(Q(61)K). The reaction between RACK1 and syndecan-2 was sustained in the presence of mutant Ras proteins, but not the reaction between p120-GAP and syndecan-2. In the presence of syndecan-2, GTP-K(B)-Ras(Q(61)K) exhibited sufficient reactivity with p120-GAP to discontinue the reaction between p120-GAP and syndecan-2. But the interference of mutant Ras disappeared when Src tyrosine kinase was introduced to stabilize the syndecan-2/p120-GAP complex. On the other hand, in the absence of syndecan-2, GTP-K(B)-Ras(Q(61)K) was found to react with RACK1. The reaction between GTP-K(B)-Ras(Q(61)K) and RACK1 could provide a mechanism to deprive RACK1 for the organization of syndecan-2/RACK1 complex and to facilitate the formation of syndecan-2/p120-GAP complex, as well as to provide docking sites for Src signaling upon transformation with oncogenic ras.

Animals↗

PubMed2005

Dimerize RACK1 upon transformation with oncogenic ras.

From our previous studies, we learned that syndecan-2/p120-GAP complex provided docking site for Src to prosecute tyrosine kinase activity upon transformation with oncogenic ras. And, RACK1 protein was reactive with syndecan-2 to keep Src inactivated, but not when Ras was overexpressed. In the present study, we characterized the reaction between RACK1 protein and Ras. RACK1 was isolated from BALB/3T3 cells transfected with plasmids pcDNA3.1-[S-ras(Q61K)] of shrimp Penaeus japonicus and RACK1 was revealed to react with GTP-K(B)-Ras(Q61K), not GDP-K(B)-Ras(Q61K). This selective interaction between RACK1 and GTP-K(B)-Ras(Q61K) was further confirmed with RACK1 of human placenta and mouse RACK1-encoded fusion protein. We found that RACK1 was dimerized upon reaction with GTP-K(B)-Ras(Q61K), as well as with 14-3-3beta and geranylgeranyl pyrophosphate, as revealed by phosphorylation with Src tyrosine kinase. We reported the complex of RACK1/GTP-K(B)-Ras(Q61K) reacted selectively with p120-GAP. This interaction was sufficient to dissemble RACK1 into monomers, a preferred form to compete for the binding of syndecan-2. These data indicate that the reaction of GTP-K(B)-Ras(Q61K) with RACK1 in dimers may operate a mechanism to deplete RACK1 from reaction with syndecan-2 upon transformation by oncogenic ras and the RACK1/GTP-Ras complex may provide a route to react with p120-GAP and recycle monomeric RACK1 to syndecan-2.

Animals↗

PubMed2005

P120-GAP associated with syndecan-2 to function as an active switch signal for Src upon transformation with oncogenic ras.

BALB/3T3 cells transfected with plasmids pcDNA3.1-[S-ras(Q(61)K)] of shrimp Penaeus japonicus were applied to reveal a complex of p120-GAP/syndecan-2 being highly expressed upon transformation. Of interest, most of the p120-GAP/syndecan-2 complex was localized at caveolae, a membrane microdomain enriched with caveolin-1. To confirm the molecular interaction between syndecan-2 and p120-GAP, we further purified p120-GAP protein from mouse brains by using an affinity column of HiTrap-RACK1 and expressed mouse RACK1-encoded fusion protein and mouse syndecan-2-encoded fusion protein in bacteria. We report molecular affinities exist between p120-GAP and RACK1, syndecan-2 and RACK1 as well as p120-GAP and syndecan-2. The selective affinity between p120-GAP and syndecan-2 was found to be sufficient to detach RACK1. The p120-GAP/syndecan-2 complex was demonstrated to keep Src tyrosine kinase in an activated form. On the other hand, the syndecan-2/RACK1 complex was found to have Src in an inactivated form. These data indicate that the p120-GAP/syndecan-2 complex at caveolae could provide a docking site for Src to transmit tyrosine signaling, implying that syndecan-2/p120-GAP functions as a tumor promoter upon transformation with oncogenic ras of shrimp P. japonicus.

Animals↗

PubMed2004

Differential effects of prenyl pyrophosphates on the phosphatase activity of phosphotyrosyl protein phosphatase.

Phosphotyrosyl protein phosphatase (PTPase) 1B was purified from human placenta. Immunoprecipitation analysis revealed that the isolated PTPase 1B appears as a complex with the receptor for protein kinase C (RACK1) and protein kinase C (PKC)delta. The abilities of PTPase 1B and PKCdelta to associate with RACK1 were reconfirmed by an in vitro reconstitution experiment. The E. coli expressed and biotinylated mice-RACK1-encoded fusion protein was capable of recruiting PTPase 1B and PKCdelta in the antibiotin immunoprecipitate as a complex of PTPase 1B/RACK1/PKCdelta. Thus PTPase 1B enzyme preparation was subjected to further purification by selective binding of PTPase 1B onto PEP(Taxol) affinity column in the absence of ATP. The purified PTPase 1B enzyme exihibited dose-dependent phosphatase activity towards [gamma-(32)P]-ATP labeled mice beta-tubulin-encoded fusion protein. The dephosphorylation reaction with PTPase 1B was enhanced with geranylgeranyl pyrophosphate, but not with farnesyl pyrophosphate. Interestingly, additional incubation of the purified PTPase 1B enzyme preparation with RACK1, geranylgeranyl pyrophosphate failed to modulate the dephosphorylation activity of PTPase 1B. In contrast, the enhancement effect of farnesyl pyrophosphate on the kinase activity of PKCdelta was sustained in the presence of RACK1. That is, farnesyl pyrophosphate may function as a signal to induce the kinase activity of PKCdelta in PTPase 1B/RACK1/PKCdelta complex but geranylgeranyl pyrophosphate may not for PTPase 1B. J. Exp. Zool. 301A:307-316, 2004.

Animals↗

PubMed2003

Farnesyl pyrophosphate promotes and is essential for the binding of RACK1 with beta-tubulin.

Receptors for activated C kinase (RACKs) are a group of protein kinase C (PKC) binding proteins that have been shown to be crucial in the translocation and subsequent functioning of PKC on activation. RACK1 isolated from BALB/3T3 cells transformed with S-ras(Q61K) exhibits receptor activity for PKCgamma as competent as that of RACK1 from BALB/3T3 cells without transformation. However, the ability of RACK1 from transformed cells to bind with beta-tubulin peptide specific for Taxol (PEPtaxol) is defective. Interestingly, when farnesyl pyrophosphate was added at the submicrogram level, the association between RACK1 and PEPtaxol was enhanced significantly in a dosage-dependent manner. A parallel finding for the enhanced effect of farnesyl pyrophosphate on tubulin binding was established with mice RACK1 expressed in vitro. On the other hand, geranylgeranyl pyrophosphate, and retinoic acid failed to modulate the binding between RACK1 and tubulin. The dissociation of RACK1 and tubulin was not effective at damaging the binding between RACK1 and membrane receptor integrin beta1 in transformed cells. These findings indicate that depletion of farnesyl pyrophosphate provides a mechanism to seal PKC signaling on the membrane with immobile RACK1 and to divert cells to aberrant growth, such as transformation.

3T3 Cells↗

PubMed2003

Effects of prenyl pyrophosphates on the binding of PKCgamma with RACK1.

Receptors for activated C kinase (RACKs) are a group of PKC binding proteins that have been shown to mediate isoform-selective functions of PKC and to be crucial in the translocation and subsequent functioning of the PKC isoenzymes on activation. RACK1 cDNA from the shrimp Penaeus japonicus was isolated by homology cloning. The hepatopancreas cDNA from this shrimp was found to encode a 318-residue polypeptide whose predicted amino acid sequence shared 91% homology with human G(beta2)-like proteins. Expression of the cDNA of shrimp RACK1 in vitro yielded a 45-kDa polypeptide with positive reactivity toward the monoclonal antibodies against RACK1 of mammals. The shrimp RACK1 was biotinylated and used to compare the effects of geranylgeranyl pyrophosphate and farnesyl pyrophosphate on its binding with PKCgamma in anti-biotin-IgG precipitates. PKCgammas were isolated from shrimp eyes and mouse brains. Both enzyme preparations were able to inhibit taxol-induced tubulin polymerization. Interestingly, when either geranylgeranyl pyrophosphate or farnesyl pyrophosphate was reduced to the submicrogram level, the recruitment activity of RACK1 with purified PKCgamma was found to increase dramatically. The activation is especially significant for RACK1 and PKCgamma from different species. The observation implies that the deprivation of prenyl pyrophosphate might function as a signal for RACK1 to switch the binding from the conventional isoenzymes of PKC (cPKC) to the novel isoenzymes of PKC (nPKC). A hydrophobic binding pocket for geranylgeranyl pyrophosphate in RACK1 is further revealed via prenylation with protein geranylgeranyl transferase I of shrimp P. japonicus.

Amino Acid Sequence↗

PubMed2002

Effects of prenyl pyrophosphates on the binding of S-Ras proteins with KSR.

BALB/3T3 cells were transformed by transfection with DNA encoding the mutated ras(Q(61)K) from shrimp Penaeus japonicus (Huang et al., 2001. J. Exp. Zool. 289:441-448). On a Western blot, the kinase suppressor of Ras (KSR) in the membrane fraction was expressed at slightly reduced level as compared to that of the untransformed cells. To understand this in more detail, the interaction of the bacterially expressed shrimp Ras (S-Ras) with KSR was investigated using KSR purified from mice brains. SDS-polyacrylamide gel electrophoresis and Western blot analysis revealed that the monomers of the purified KSR have a relative molecular mass of 60,000. Purified KSR was found to bind with digoxigenylated S-ras-encoding fusion protein (Dig-S-Ras) with high affinity in the absence of ATP, and the binding activity of KSR was sustained upon phosphorylation of Dig-S-Ras with mitogen-activated protein kinase (MAPK). The association of purified KSR with S-Ras was confirmed. Differences between the effects of farnesyl pyrophosphate and geranylgeranyl pyrophosphate on the binding of S-Ras with the purified KSR were assessed. Densitometer analysis revealed that at nanogram concentration, farnesyl pyrophosphate inhibited the binding of S-Ras with KSR competently, but geranylgeranyl pyrophosphate did not. The present study provides the evidence that decrease of the concentration of farnesyl pyrophosphate to sub-microgram levels lower the affinity of Ras proteins with KSR in the signaling pathway.

3T3 Cells↗

PubMed2002

Selective enhanced phosphorylation of shrimp beta-tubulin by PKC-delta with PEP(taxol), a synthetic peptide encoding the taxol binding region.

Beta-tubulin cDNA from the shrimp Penaeus japonicus was isolated by homology cloning. Expression of cDNA in Escherichia coli yielded a 55 kDa polypeptide, positive for monoclonal antibodies against mammalian beta-tubulin. Autoradiography demonstrated the bacterially expressed hepatopancreas beta-tubulin of P. japonicus is specifically phosphorylated by the delta isoenzyme of protein kinase C (PKC-delta) purified from the plasma membrane of the shrimp heart, in the presence of the receptor for activated PKC (RACK), but not in its absence. Purified shrimp heart PKC-delta is able to phosphorylate bacterially expressed shrimp beta-tubulin without the presence of Ca(++), but requires Mg(++). The kinase activity of purified PKC-delta on bacterially expressed beta-tubulin was enhanced by incubation with PEP(taxol), a synthetic peptide encoding the taxol-binding region of beta-tubulin. In other words, PEP(taxol) modulates the kinase activity of PKC-delta through RACK.

Amino Acid Sequence↗