Development and characterization of potent and specific peptide inhibitors of p60<sup>c‐src</sup> protein tyrosine kinase using pseudosubstrate‐based inhibitor design approach

Kamath, J. R.; Liu, Ruiwu; Enstrom, Amanda; Lou, Qiang; Lam, Kit S.

doi:10.1046/j.1399-3011.2003.00094.x

Cited by 17 publications

(17 citation statements)

References 44 publications

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“…31). Target specificity of these peptides has been shown earlier (31). Both peptides reduced Id1 transcript levels in a dose-dependent manner.…”

Section: Resultsmentioning

confidence: 53%

“…The Src-blocking peptides CpraYKYY-hAla-r7 and CpraYKYY-hAla-k7 (provided by Dr. Kit Lam, University of California Davis Cancer Center, Sacramento, CA) were solubilized in sterile H 2 O to obtain a 25 mmol/L stock solution (31). Recombinant human BMP-2 (R&D Systems) was reconstituted in 4 nmol/L HCl containing 0.1% bovine serum albumin (BSA) to obtain a 10 Ag/mL stock solution.…”

Section: Methodsmentioning

confidence: 99%

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Regulation of Id1 Expression by Src: Implications for Targeting of the Bone Morphogenetic Protein Pathway in Cancer

et al. 2008

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Deregulated activation of the Src tyrosine kinase and heightened Id1 expression are independent mediators of aggressive tumor biology. The present report implicates Src signaling as a critical regulator of Id1 gene expression. Microarray analyses showed that Id family genes were among the most highly down-regulated by incubation of A549 lung carcinoma cells with the small-molecule Src inhibitor AZD0530. Id1 transcript and protein levels were potently reduced in a dose-dependent manner concomitantly with the reduction of activated Src levels. These effects were conserved across a panel of lung, breast, prostate, and colon cancer cell lines and confirmed by the ability of PP2, Src siRNA, and Srcblocking peptides to suppress Id1 expression. PP2, AZD0530, and dominant-negative Src abrogated Id1 promoter activity, which was induced by constitutively active Src. The Srcresponsive region of the Id1 promoter was mapped to a region 1,199 to 1,360 bps upstream of the translation start site and contained a Smad-binding element. Src was also required for bone morphogenetic protein-2 (BMP-2)-induced Id1 expression and promoter activity, was moderately activated by BMP-2, and complexed with Smad1/5. Conversely, Src inhibitors blocked Smad1/5 nuclear translocation and binding to the Src-responsive region of the Id1 promoter. Consistent with a role for Src and Id1 in cancer cell invasion, Src inhibitors and Id1 siRNA decreased cancer cell invasion, which was increased by Id1 overexpression. Taken together, these results reveal that Src positively interacts with the BMP-Smad-Id pathway and provide new ways for targeted inhibition of Id1. [Cancer Res 2008;68(7):2250-8]

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“…31). Target specificity of these peptides has been shown earlier (31). Both peptides reduced Id1 transcript levels in a dose-dependent manner.…”

Section: Resultsmentioning

confidence: 53%

Section: Methodsmentioning

confidence: 99%

Regulation of Id1 Expression by Src: Implications for Targeting of the Bone Morphogenetic Protein Pathway in Cancer

et al. 2008

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“…The second group includes peptide inhibitors modified from known substrates. These include nonphosphorylatable substrate-based peptides that inhibit protein kinase C (37), calmodulin-dependent protein kinase II (38), MAPK-activated protein kinase-2 (39), Akt/protein kinase B (40), p60 c-Src protein-tyrosine kinase (41) and ZAP-70 tyrosine kinase (42). Inhibitors from both of these groups act as pseudosubstrates for the relevant kinases.…”

Section: Discussionmentioning

confidence: 99%

The Critical Features and the Mechanism of Inhibition of a Kinase Interaction Motif-based Peptide Inhibitor of JNK

Barr

Boehm

Attwood

et al. 2004

Journal of Biological Chemistry

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We previously reported that a small peptide based on amino acids 143-153 of the c-Jun N-terminal kinase (JNK)-binding domain of JIP-1 functioned as an in vitro inhibitor of JNK activity. This peptide (TI-JIP: RP-KRPTTLNLF) resembles the kinase-interaction motif (KIM ‫؍‬ (K/R) 2-3 X 1-6 (L/I)X(L/I)), which is common to upstream activators, downstream substrates, phosphatases, and scaffold proteins present in MAPK cascades. In this study, we characterized the mechanism of JNK inhibition by this peptide and further investigated the biochemical features of this peptide resulting in potent JNK inhibition. We also tested various KIM-based peptides for their ability to inhibit JNK activity. TI-JIP was found to be competitive with respect to the phosphoacceptor substrate c-Jun (K I ‫؍‬ 0.39 ؎ 0.08 M), and exhibit mixed (non-competitive) inhibition with respect to ATP. All seven substitutions of Pro-5 we tested significantly reduced the JNK inhibition, as did altering the Pro-5 to Leu-8 spacing. When we independently tested eight substitutions of either Thr-6 or Thr-7, only one substitution in each position was well tolerated. Furthermore, peptides based on the KIMs from other proteins were significantly less potent JNK inhibitors than TI-JIP, including a peptide from the JNK interactor Sab that contained all critical inhibitory residues present in TI-JIP. Therefore, despite having previously identified Arg-4, Pro-5, Leu-8, and Leu-10 in TI-JIP as independently critical for mediating JNK inhibition, we find their presence in other 11-mer peptides is not sufficient for JNK inhibition. TI-JIP is therefore a unique KIM-based inhibitor of JNK activity.

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“…The LRT-SIFP consists of a pseudosubstratebased peptide inhibitor of SFK (MIYKYYF; ref. 25), YFP, a flag tag, and a lipid raft-targeting sequence (Fig. 3A).…”

Section: Tm-srcus Can Monitor Sfk Activation Induced By a Physiologicmentioning

confidence: 99%

“…LRT-SIFP contains the pseudosubstrate-based peptide inhibitor that is preferentially phosphorylated by activated SFK to inhibit the enzymatic activity of SFK (25). We examined whether LRT-SIFP was phosphorylated by SFK in MCF-7 cells.…”

Section: Tm-srcus Can Monitor Sfk Activation Induced By a Physiologicmentioning

confidence: 99%

Lipid Raft–Specific Knockdown of Src Family Kinase Activity Inhibits Cell Adhesion and Cell Cycle Progression of Breast Cancer Cells

et al. 2007

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Src family kinase (SFK) is known to control various cell functions, but the significance of the location of its activation was largely unknown. We herein revealed that SFK activation occurs in lipid rafts. Based on this finding, we have developed a lipid raft-targeted SFK inhibitory fusion protein (LRT-SIFP) that inhibits the SFK activity in lipid rafts. LRT-SIFP has a peptide inhibitor of SFK and a lipid raft-targeting sequence in which two cysteine residues are palmitoylated for clustering in lipid rafts. LRT-SIFP was found to inhibit cell adhesion and cell cycle progression of human breast cancer cell lines MCF-7 and MDA-MB231. On the other hand, the cell functions of MCF-7 cells were found to be not affected with a previously developed peptide inhibitor of SFK that lacks the lipid rafttargeting sequence. In addition, when we replaced the targeting sequence of LRT-SIFP with the consensus sequence for geranylgeranylation to make LRT-SIFP unable to cluster in lipid rafts, this mutated LRT-SIFP did not show any effect on the above cell functions of MCF-7 cells. Furthermore, in contrast to the breast cancer cell lines, LRT-SIFP did not show any inhibitory effect on cell adhesion and cell cycle progression of human normal cell line HEK293. The present lipid raft-specific knockdown of SFK activity would potentially be useful for selective cancer therapy to prevent tumorigenesis and metastasis of breast cancer cells. [Cancer Res 2007;67(17):8139-48]

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Development and characterization of potent and specific peptide inhibitors of p60^c‐src protein tyrosine kinase using pseudosubstrate‐based inhibitor design approach

Cited by 17 publications

References 44 publications

Regulation of Id1 Expression by Src: Implications for Targeting of the Bone Morphogenetic Protein Pathway in Cancer

Regulation of Id1 Expression by Src: Implications for Targeting of the Bone Morphogenetic Protein Pathway in Cancer

The Critical Features and the Mechanism of Inhibition of a Kinase Interaction Motif-based Peptide Inhibitor of JNK

Lipid Raft–Specific Knockdown of Src Family Kinase Activity Inhibits Cell Adhesion and Cell Cycle Progression of Breast Cancer Cells

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Development and characterization of potent and specific peptide inhibitors of p60c‐src protein tyrosine kinase using pseudosubstrate‐based inhibitor design approach

Cited by 17 publications

References 44 publications

Regulation of Id1 Expression by Src: Implications for Targeting of the Bone Morphogenetic Protein Pathway in Cancer

Regulation of Id1 Expression by Src: Implications for Targeting of the Bone Morphogenetic Protein Pathway in Cancer

The Critical Features and the Mechanism of Inhibition of a Kinase Interaction Motif-based Peptide Inhibitor of JNK

Lipid Raft–Specific Knockdown of Src Family Kinase Activity Inhibits Cell Adhesion and Cell Cycle Progression of Breast Cancer Cells

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Development and characterization of potent and specific peptide inhibitors of p60^c‐src protein tyrosine kinase using pseudosubstrate‐based inhibitor design approach