Exit from mitosis in budding yeast requires a group of essential proteins--including the GTPase Tem1 and the protein phosphatase Cdc14--that downregulate cyclin-dependent kinase activity. We identified a mutation, net1-1, that bypasses the lethality of tem1 delta. NET1 encodes a novel protein, and mass spectrometric analysis reveals that it is a key component of a multifunctional complex, denoted RENT (for regulator of nucleolar silencing and telophase), that also contains Cdc14 and the silencing regulator Sir2. From G1 through anaphase, RENT localizes to the nucleolus, and Cdc14 activity is inhibited by Net1. In late anaphase, Cdc14 dissociates from RENT, disperses throughout the cell in a Tem1-dependent manner, and ultimately triggers mitotic exit. Nucleolar sequestration may be a general mechanism for the regulation of diverse biological processes.

Voltage-sensitive sodium channels are responsible for the initiation and propagation of the action potential and therefore are important for neuronal excitability. Complementary DNA clones encoding the beta 1 subunit of the rat brain sodium channel were isolated by a combination of polymerase chain reaction and library screening techniques. The deduced primary structure indicates that the beta 1 subunit is a 22,851-dalton protein that contains a single putative transmembrane domain and four potential extracellular N-linked glycosylation sites, consistent with biochemical data. Northern blot analysis reveals a 1,400-nucleotide messenger RNA in rat brain, heart, skeletal muscle, and spinal cord. Coexpression of beta 1 subunits with alpha subunits increases the size of the peak sodium current, accelerates its inactivation, and shifts the voltage dependence of inactivation to more negative membrane potentials. These results indicate that the beta 1 subunit is crucial in the assembly, expression, and functional modulation of the heterotrimeric complex of the rat brain sodium channel.

Protein tyrosine phosphatases (PTPs) represent a diverse family of enzymes that exist as integral membrane and nonreceptor forms. The PTPs, with specific activities in vitro 10 to 1000 times greater than those of the protein tyrosine kinases would be expected to effectively control the amount of phosphotyrosine in the cell. They dephosphorylate tyrosyl residues in vivo and take part in signal transduction and cell cycle regulation. Most of the transmembrane forms, such as the leukocyte common antigen (CD45), contain two conserved intracellular catalytic domains; but their external segments are highly variable. The structural features of the transmembrane forms suggest that these receptor-linked PTPs are capable of transducing external signals; however, the ligands remain unidentified. A hypothesis is proposed explaining how phosphatases might act synergistically with the kinases to elicit a full physiological response, without regard to the state of phosphorylation of the target proteins.

It has been proposed on the basis of amino acid sequence homology that the leukocyte common antigen CD45 represents a family of catalytically active, receptor-linked protein tyrosine phosphatases [Charbonneau, H., Tonks, N. K., Walsh, K. A., & Fischer, E. H. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7182-7186]. The present study confirms that CD45 possesses intrinsic protein tyrosine phosphatase (PTPase) activity. First, a mouse monoclonal antibody to CD45 (mAb 9.4) specifically eliminated, by precipitation, PTPase activity from a high Mr fraction containing CD45, prepared by gel filtration (Sephacryl S200) of a Triton X-100 extract of human spleen. Second, PTPase activity was demonstrated in a highly purified preparation of CD45 that was eluted with a high pH buffer from an affinity column, constructed from the same antibody. Third, on sucrose density gradient centrifugation, PTPase activity was only found in those fractions that contained CD45 as determined by Western analysis. When CD45 was caused to aggregate, first by reacting it with mAb 9.4 and then adding a secondary, cross-linking anti-mouse mAb, the PTPase activity shifted to the same higher Mr fractions that contained CD45. No shift in CD45 or PTPase was observed following addition of a control IgG2a. On this basis, it is concluded that CD45 is a protein tyrosine phosphatase.

Calcium can function as a second messenger through stimulation of calcium-dependent protein kinases. A protein kinase that requires calcium but not calmodulin or phospholipids for activity has been purified from soybean. The kinase itself binds calcium with high affinity. A complementary DNA clone for this kinase has been identified; it encodes a protein with a predicted molecular mass of 57,175 daltons. This protein contains a catalytic domain similar to that of calmodulin-dependent kinases and a calmodulin-like region with four calcium binding domains (EF hands). The predicted structure of this kinase explains its direct regulation via calcium binding and establishes it as a prototype for a new family of calcium-regulated protein kinases.