Hiromi Fukamachi

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 ∼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 (K D = 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 β-sheets of the PH domains of known tertiary structures. On the other hand, the C1 regulatory region of PKCε 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-α-PMA) was ineffective. The ζ 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 PKCζ 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 β2–β3 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.

Granulocyte colony-stimulating factor(G-CSF) receptors on acute myeloblastic leukaemia cells and their relationship with the proliferative response to G-CSF in clonogenic assay

Summary. The number and the affinity of granulocyte colony‐stimulating factor (G‐CSF) receptors expressed by blast cells in acute myeloblastic leukaemia (AML) were determined using radiolabeled recombinant human G‐CSF (rhG‐CSF). Eighteen of 20 patients demonstrated specific binding, and Scatchard analysis revealed a single class of high affinity (Kd 15‐130 pM) G‐CSF receptors on the AML blasts. The number of G‐CSF receptors varied from 55 to 1200 per cell (mean 278). In the remaining two patients, specific binding was not observed. The number of G‐CSF receptors did not differ significantly between various AML subtypes, but the mean receptor number was the highest on type M2 blasts. A chemical cross‐linking study revealed that the G‐CSF receptor has an approximate molecular weight of 140 000. Autoradiography showed heterogeneity of the distribution of G‐CSF receptors on the AML blasts obtained from a single patient. The number of colonies stimulated by the addition of rhG‐CSF varied from 0 to 566 per dish, and blast colony formation was observed in eight of 20 patients. The population mean of G‐CSF receptor number expressed by blasts that formed colonies on stimulation with rhG‐CSF was significantly higher than that on blasts which did not form colonies. These results suggest that a proliferative response of AML blasts to G‐CSF may be predicted when the blasts express a large number of G‐CSF receptors. Accordingly, it may be safer to restrict the clinical use of G‐CSF to AML patients who have blasts with a low G‐CSF receptor expression and no response to G‐CSF in blast colony assay.

Activation of Multiple Protein Kinases Including a MAP Kinase upon FcεRI Cross-Linking

Previous studies have shown that protein-serine/threonine kinases and protein-tyrosine kinase(s) are activated by cross-linking of the high-affinity receptor for IgE, FceRI, on mast cells and basophil

Mutant protein of recombinant human granulocyte colony-stimulating factor for receptor binding assay

A new mutant protein of recombinant human granulocyte colony-stimulating factor (rhG-CSF) was produced for the studies on receptors for human G-CSF. The mutant protein [(Tyr1, Tyr3]rhG-CSF), the biological activity of which was almost equal to that of rhG-CSF, was prepared by the replacement of threonine-1 and leucine-3 of rhG-CSF with tyrosine. The radioiodinated preparation of the mutant protein showed high specific radioactivity and retained full biological activity for at least 3 weeks. The binding capacity of the radioiodinated ligand was compared with that of [35S]rhG-CSF. Both radiolabeled ligands showed specific binding to murine bone marrow cells. Unlabeled rhG-CSF and human G-CSF purified from the culture supernatant of the human bladder carcinoma cell line 5637 equally competed for the binding of labeled rhG-CSFs in a dose-dependent manner, demonstrating that the sugar moiety of human G-CSF made no contribution to the binding of human G-CSF to target cells. In contrast, all other colony-stimulating factors and lymphokines examined did not affect the binding. Scatchard analysis of the specific binding of both labeled ligands revealed a single class of binding site with an apparent dissociation constant (Kd) of 20-30 pM and 100-200 maximal binding sites per cell. These data indicate that the radioiodinated preparation of the mutant protein binds the same specific receptor with the same affinity as [35S]rhG-CSF. The labeled mutant protein also showed specific binding to human circulating neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of a chymotrypsin-like inhibitor, TPCK, on histamine release from cultured human mast cells

The involvement of endogenous proteases in the secretory process from human mast cells remains to be clarified.