Mammalian CAP interacts with CAP, CAP2, and actin

Abstract

We previously identified human CAP, a homolog of the yeast adenylyl cyclase-associated protein. Previous studies suggest that the N-terminal and C-terminal domains of CAP have distinct functions. We have explored the interactions of human CAP with various proteins. First, by performing yeast two-hybrid screens, we have identified peptides from several proteins that interact with the C-terminal and/or the N-terminal domains of human CAP. These peptides include regions derived from CAP and BAT3, a protein with unknown function. We have further shown that MBP fusions with these peptides can associate in vitro with the N-terminal or C-terminal domains of CAP fused to GST. Our observations indicate that CAP contains regions in both the N-terminal and C-terminal domains that are capable of interacting with each other or with themselves. Furthermore, we found that myc-epitope-tagged CAP coimmunoprecipitates with HA-epitope-tagged CAP from either yeast or mammalian cell extracts. Similar results demonstrate that human CAP can also interact with human CAP2. We also show that human CAP interacts with actin, both by the yeast two-hybrid test and by coimmunoprecipitation of epitope-tagged CAP from yeast or mammalian cell extracts. This interaction requires the C-terminal domain of CAP, but not the N-terminal domain. Thus CAP appears to be capable of interacting in vivo with other CAP molecules, CAP2, and actin. We also show that actin co-immunoprecipitates with HA-CAP2 from mammalian cell extracts.

Three Yeast Proteins Related to the Human Candidate Tumor Suppressor p33ING1 Are Associated with Histone Acetyltransferase Activities

“…pLexA-⌬PHD and pLEXA-PHD were generated by inserting codons 1 to 222 and 222 to 282, respectively, of YNG2 into pBTM116. pADHA-Yng2, pADHA-Png1, and pADHA-Ing1 were generated by cloning the PCR-derived open reading frame (ORF) of YNG2, S. pombe PNG1, or human p33 ING1 respectively, into pAD4.H, which contains the 2m origin of replication and ADH1 promoter (32). YEp-PDE2 contains S. cerevisiae PDE2 in YEp13, as previously described (65).…”

“…This PCR product was cloned into pADGFPHA-Yng2 as an EcoRI/SacI fragment to replace the C-terminal PHD finger of Yng2 with that of Yng1. pADmyc-Yng2 was generated by cloning the PCR-derived ORF of YNG2 in pUAD6 (32). pADmyc-Tra1 was derived by shuttling the TRA1 ORF as a NotI(filled-in)/SacI fragment from p1259 (a kind gift from C. Brandl) into the SmaI/SacI sites of a modified pUAD6 (SalI digested, filled in, and religated).…”

The role of cyclase-associated protein in regulating actin filament dynamics – more than a monomer-sequestration factor

“…1C). Furthermore, a study, in which short CAP fragments where used, suggests that the N-terminal part of CAP can bind to both the N-terminal and C-terminal sections of a second CAP (Hubberstey et al, 1996). A mutational study of Dictyostelium CAP suggests that several configurations of a CAP dimer are possible (Yusof et al, 2006).…”

Overexpression of Cyclase-Associated Protein 2 in Multistage Hepatocarcinogenesis

“…CAP also binds monomeric actin and, therefore, also possesses a cytoskeletal function (11 -15). However, only a few studies about CAP have previously been reported in mammalian cells (16) and no studies about CAP in human cancer have been published. In the present study, we have investigated the expression of CAP2 and proved its overexpression in multistage carcinogenesis in HCC.…”