Interactions between protein kinase C and pleckstrin homology domains. Inhibition by phosphatidylinositol 4,5-bisphosphate and phorbol 12-myristate 13-acetate.
Pleckstrin homology (PH) domains comprised of loosely conserved sequences of approximately 100 amino acid residues are a functional protein motif found in many signal-transducing and cytoskeletal proteins. We recently demonstrated that the PH domains of Tec family protein-tyrosine kinases Btk and Emt (equal to Itk and Tsk) interact with protein kinase C (PKC) and that PKC down-regulates Btk by phosphorylation. In this study we have characterized the PKC-BtkPH domain interaction in detail. Using pure PKC preparations, it was shown that the Btk PH domain interacts with PKC with high affinity (KD = 39 nM). Unlike other tested phospholipids, phosphatidylinositol 4,5-bisphosphate, which binds to several PH domains, competed with PKC for binding to the PH domain apparently because their binding sites on the amino-terminal portion of the PH domains overlap. The minimal PKC-binding sequence within the Btk PH domain was found to correspond roughly to the second and third beta-sheets of the PH domains of known tertiary structures. On the other hand, the C1 regulatory region of PKCepsilon containing the pseudosubstrate and zinc finger-like sequences was found to be sufficient for strong binding to the Btk PH domain. Phorbol 12-myristate 13-acetate (PMA), a potent activator of PKC that interacts with the C1 region of PKC, inhibited the PKC-PH domain interaction, whereas the bioinactive PMA (4-alpha-PMA) was ineffective. The zeta isoform of PKC, which has a single zinc finger-like motif instead of the two tandem zinc finger-like sequences present in conventional and novel PKC isoforms, does not bind PMA. Thus, as expected, PH domain binding with PKCzeta was not interfered with by PMA. Further, inhibitors that are known to attack the catalytic domains of serine/threonine kinases did not affect this PKC-PH domain interaction. In contrast, the presence of physiological concentrations of Ca2+ induced less than a 2-fold increase in PKC-PH domain binding. These results indicate that PKC binding to PH domains involve the beta2-beta3 region of the Btk PH domain and the C1 region of PKC, and agents that interact with either of these regions (i.e. phosphatidylinositol 4,5-bisphosphate binding to the PH domain and PMA binding to the C1 region of PKC) might act to regulate PKC-PH domain binding.
Yao
L
L
Division of Allergy, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, USA.
Suzuki
H
H
Ozawa
K
K
Deng
J
J
Lehel
C
C
Fukamachi
H
H
Anderson
W B
WB
Kawakami
Y
Y
Kawakami
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010750:Phosphoproteins; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D016415:Sequence Alignment; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism
D001798:Blood Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D010750:Phosphoproteins; C039077:platelet protein P47; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate