Gernot Maier

Stimulation of phospholipase C-beta2 by the Rho GTPases Cdc42Hs and Rac1.

Neutrophils contain a soluble guanine‐nucleotidebinding protein, made up of two components with molecular masses of 23 and 26 kDa, that mediates stimulation of phospholipase C‐β2 (PLCβ2). We have identified the two components of the stimulatory heterodimer by amino acid sequencing as a Rho GTPase and the Rho guanine nucleotide dissociation inhibitor LyGDI. Using recombinant Rho GTPases and LyGDI, we demonstrate that PLCβ2 is stimulated by guanosine 5′‐O‐(3‐thiotriphosphate) (GTP[S])‐activated Cdc42Hs×LyGDI, but not by RhoA×LyGDI. Stimulation of PLCβ2, which was also observed for GTP[S]‐activated recombinant Rac1, was independent of LyGDI, but required C‐terminal processing of Cdc42Hs/Rac1. Cdc42Hs/Rac1 also stimulated PLCβ2 in a system made up of purified recombinant proteins, suggesting that this function is mediated by direct protein–protein interaction. The Cdc42Hs mutants F37A and Y40C failed to stimulate PLCβ2, indicating that the Cdc42Hs effector site is involved in this interaction. The results identify PLCβ2 as a novel effector of the Rho GTPases Cdc42Hs and Rac1, and as the first mammalian effector directly regulated by both heterotrimeric and low‐molecular‐mass GTP‐binding proteins.

Cytokeratin domains involved in heterotypic complex formation determined by in-vitro binding assays☆

Cytokeratins are constituent proteins of intermediate filaments (IFs) that form heterotypic tetrameric IF subunits containing two polypeptide chains of each of the two cytokeratin subfamilies, i.e. the acidic (type I) and the basic (type II). To locate the molecular domains involved in the formation of these heterotypic complexes, we have developed a binding assay in which total cellular or cytoskeletal polypeptides, or proteolytically prepared cytokeratin fragments, are separated by one-, or two-dimensional gel electrophoresis, blot-transferred on to nitrocellulose paper and probed with radio-iodinated purified cytokeratin polypeptides or fragments thereof, using buffers of various ionic strengths with or without 4 M-urea. Using these polypeptides in the binding assay, specific heterotypic binding was observed between complementary cytokeratin polypeptides of the two subfamilies (but not with other IF proteins) and between the corresponding alpha-helical rod domain fragments. Both rod coils 1 and 2 of the type II cytokeratin 8 bound to the rod (coils 1 and 2) fragment of type I cytokeratins, and this binding occurred at both low and high ionic strengths. The results obtained indicate that: (1) the binding between cytokeratin polypeptides of the complementary type is stronger and more selective than interactions of cytokeratins with other IF and non-IF proteins; (2) both the head and the tail portions of the proteins are not required for heterotypic complex formation; (3) the complementarity information located in the alpha-helical portions of the rod domain, and in short sequences immediately flanking them, is sufficient to discriminate between the two types of cytokeratins and to secure the formation of heterotypic cytokeratin complexes; (4) both coils 1 and 2 of the rod can contribute to this association; and (5) the formation of the heterotypic cytokeratin complex is not critically dependent upon ionic interactions. Our results are further compatible with the concept that the heterotypic binding takes place between cytokeratin homodimer coiled-coils.

Human Supt5h protein, a putative modulator of chromatin structure, is reversibly phosphorylated in mitosis

The Saccharomyces cerevisiae proteins Spt4p, Spt5p and Spt6p are involved in transcriptional repression by modulating the structure of chromatin. From HeLa cells we have purified a human homologue of Spt5p, Supt5hp, and show here that the protein is reversibly phosphorylated in mitosis. The cloned cDNA predicts a protein of 1087 residues with 31% identity to yeast Spt5p. It includes an acidic N‐terminus, a putative nuclear localization signal and a C‐terminal region containing two different repeated motifs. One of them, with the consensus sequence P‐T/S‐P‐S‐P‐Q/A‐S/G‐Y, is similar to the C‐terminal domain in the largest subunit of RNA polymerase II.

A sequence variant in the N-terminal region of the catalytic subunit of the cAMP-dependent protein kinase

Protein kinase; Cyclic AMP; (Bovine)

Use of a Metalloproteinase Specific for the Amino Side of Asp in Protein Sequencing

A larger panel of proteolytic enzymes analogous to DNA restriction enzymes would be useful for analyzing protein structures. As we are sequencing larger polypeptides in smaller quantities, generation of a moderate number of fragments would, in most cases, facilitate their separation and overlapping. In addition, such proteinases would be helpful in the elucidation of domains with biological activity, in generating peptide maps [1] and in the recent attempts to sequence end-labeled proteins on gels [2, 3].

Cell density dependent DNA replication in Ehrlich ascites tumour cells

DNA replication of Ehrlich ascites tumour cells was investigated in suspensions with different cell densities by incorporation in vitro of tritiated thymidine and alkaline sucrose gradient analysis of the newly formed DNA. It is demonstrated that the incorporation of [3H]thymidine and chain growth of newly made DNA decreases with increasing cell density. The inhibition of DNA synthesis observed at high cell densities can be prevented if diffusible substances are removed by incubating the cells in dialysis tubes. This indicates that the changes in DNA synthesis are caused by diffusible inhibitors released from the tumour cells.

Characterization of Proteins Involved in Mammalian Mitosis

Our interest in the molecular mechanisms of mitosis includes aspects of the mechanochemistry and fidelity of chromosome distribution, and the control of ontogeny which, in addition to differentiation, requires specific timing and positioning of cell divisions to generate the pattern of some 1014 cells of a mammal from a single fertilized egg and to compensate for cell losses.

Generation of Starting Points for Microsequencing with a Protease Specific for the Amino Side of Aspartyl Residues

To obtain maximum sequence information from minute amounts of protein, it is desirable to perform specific and complete cleavage at a limited number of sites. To date this criterion is met by very few methods. We wish to draw attention to a metalloprotease from Pseudomonas fragi which is specific for the amino side of aspartyl residues. It has proven a valuable tool in our structural work on intracellular proteins. The cleavage pattern differs considerably from other enzymes commonly used.