Binding of elements of protein kinase C-alpha regulatory domain to lamin B1.
Previous results from our laboratory have demonstrated that lamin B1 is a protein kinase C (PKC)-binding protein. Here, we have identified the regions of PKC-alpha that are important for this binding. By means of overlay assays and fusion proteins made of glutathione-S-transferase (GST) fused to elements of the regulatory domain of rat PKC-alpha, we have established that binding occurs through both the V1 region and a portion of the C2 region (i.e., the calcium-dependent lipid binding [CaLB] domain) of the kinase. In particular, we have found that amino acids 200-217 of the CaLB domain are essential for binding lamin B1, as a synthetic peptide corresponding to this stretch of amino acids prevented the interaction between the CaLB domain of PKC-alpha and lamin B1. In agreement with the results of other investigators, we have determined that binding of regulatory elements of PKC-alpha to lamin B1 does not require the presence of cofactors such as PS and Ca(2+). We have also found that the binding site of lamin B1 for PKC-alpha is localized in the carboxyl-terminus of the lamin. Our findings may prove to be important in shedding more light on the mechanisms that regulate PKC functions within the nuclear compartment and may also lead to the synthesis of isozyme-specific pharmacological tools to attenuate or reverse PKC-dependent nuclear signalling pathways important for the pathogenesis of cancer.
Dipartimento di Morfologia Umana Normale, Università di Trieste, via Manzoni 16, 34138 Trieste, Italy.
Giovanna
G
Tabellini
Roberta
R
Bortul
Michela
M
Aluigi
Anna Maria
AM
Billi
Renato
R
Bareggi
Vittorio
V
Grill
Paola
P
Narducci
Alberto M
AM
Martelli
D016475:3T3 Cells; D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000595:Amino Acid Sequence / Q000502:physiology; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology*; D005057:Eukaryotic Cells / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D005809:Genes, Regulator / Q000502:physiology*; D005982:Glutathione Transferase; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D034921:Lamin Type B / Q000378:metabolism*; D051379:Mice; D009369:Neoplasms / Q000201:enzymology*; D009687:Nuclear Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D011485:Protein Binding / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D017434:Protein Structure, Tertiary / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D034921:Lamin Type B; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; C496628:PRKCA protein, human; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha