Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.

SUMMARY Tissue homeostasis in metazoans is regulated by transitions of cells between quiescence and proliferation. The hallmark of proliferating populations is progression through the cell cycle, which is driven by cyclin-dependent kinase (CDK) activity. Here, we introduce a live-cell sensor for CDK2 activity and unexpectedly found that proliferating cells bifurcate into two populations as they exit mitosis. Many cells immediately commit to the next cell cycle by building up CDK2 activity from an intermediate level, while other cells lack CDK2 activity and enter a transient state of quiescence. This bifurcation is directly controlled by the CDK inhibitor p21 and is regulated by mitogens during a restriction window at the end of the previous cell cycle. Thus, cells decide at the end of mitosis to either start the next cell cycle by immediately building up CDK2 activity or to enter a transient G0-like state by suppressing CDK2 activity.

The range of messenger action of a point source of Ca2+ or inositol 1,4,5-trisphosphate (IP3) was determined from measurements of their diffusion coefficients in a cytosolic extract from Xenopus laevis oocytes. The diffusion coefficient (D) of [3H]IP3 injected into an extract was 283 microns 2/s. D for Ca2+ increased from 13 to 65 microns 2/s when the free calcium concentration was raised from about 90 nM to 1 microM. The slow diffusion of Ca2+ in the physiologic concentration range results from its binding to slowly mobile or immobile buffers. The calculated effective ranges of free Ca2+ before it is buffered, buffered Ca2+, and IP3 determined from their diffusion coefficients and lifetimes were 0.1 micron, 5 microns, and 24 microns, respectively. Thus, for a transient point source of messenger in cells smaller than 20 microns, IP3 is a global messenger, whereas Ca2+ acts in restricted domains.

Deviations in basal Ca2+ levels interfere with receptor-mediated Ca2+ signaling as well as endoplasmic reticulum (ER) and mitochondrial function. While defective basal Ca2+ regulation has been linked to various diseases, the regulatory mechanism that controls basal Ca2+ is poorly understood. Here we performed an siRNA screen of the human signaling proteome to identify regulators of basal Ca2+ concentration and found STIM2 as the strongest positive regulator. In contrast to STIM1, a recently discovered signal transducer that triggers Ca2+ influx in response to receptor-mediated depletion of ER Ca2+ stores, STIM2 activated Ca2+ influx upon smaller decreases in ER Ca2+. STIM2, like STIM1, caused Ca2+ influx via activation of the plasma membrane Ca2+ channel Orai1. Our study places STIM2 at the center of a feedback module that keeps basal cytosolic and ER Ca2+ concentrations within tight limits.

Many signaling, cytoskeletal, and transport proteins have to be localized to the plasma membrane (PM) in order to carry out their function. We surveyed PM-targeting mechanisms by imaging the subcellular localization of 125 fluorescent protein-conjugated Ras, Rab, Arf, and Rho proteins. Out of 48 proteins that were PM-localized, 37 contained clusters of positively charged amino acids. To test whether these polybasic clusters bind negatively charged phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] lipids, we developed a chemical phosphatase activation method to deplete PM PI(4,5)P 2 . Unexpectedly, proteins with polybasic clusters dissociated from the PM only when both PI(4,5)P 2 and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] were depleted, arguing that both lipid second messengers jointly regulate PM targeting.Small guanosine triphosphatases (GTPases) from the Ras, Rho, Arf, and Rab subfamilies often exert their role at the PM where they control diverse signaling, cytoskeletal, and transport processes (1-3). KRas, CDC42, and other family members require a cluster of positively charged amino acids for PM localization and activity (2, 4). In vitro studies indicate that the physiological PM binding partner of such polybasic clusters could be phosphatidylserine, which has one negative charge, or the less abundant lipid second messenger PI(4,5)P 2 , which has four negative charges (5-7). We took a genomic survey approach and investigated PM-targeting mechanisms by confocal imaging of 125 cyan fluorescent protein (CFP)-tagged constitutively active small GTPases (8). Expression in NIH3T3 and HeLa cells showed that 48 small GTPases were fully or partially localized to the PM (Fig. 1A and fig. S1).Thirty-seven of these PM-localized small GTPases had C-terminal polybasic clusters consisting of four or more Lys or Arg residues at positions 5 to 20 from the C terminus ( Fig. 1B and fig. S1). Polybasic clusters were found in three forms: They were present together with N-terminal myristoylation consensus sequences (as in Arl4) (9) or with C-terminal prenylation consensus sequences (as in KRas) (5, 6, 10), or they lacked lipid modifications (as in Rit) (11). We called these three combinations polybasic-myristoyl, polybasic-prenyl, and polybasic-nonlipid PM-targeting motifs, respectively. A number of remaining PMtargeted small GTPases had a combined prenylation and palmitoylation consensus sequence that mediated PM targeting without requiring polybasic amino acids (as does that of HRas) (Fig. 1D).To test whether polybasic clusters are anchored to the PM by binding to PI(4,5)P 2 (14), we hydrolyzed PM PI(4,5)P 2 by rapid targeting of Inp54p, a 5′ specific PI(4,5)P 2 phosphatase (15), to the PM. This method is based on a PM-localized FK506-binding protein (FKBP12)-rapamycin-binding (FRB) construct and a cytosolic Inp54p enzyme conjugated with FKBP12 (CF-Inp) that can be translocated to the PM by chemical heterodimerization by using a rapamycin analog, iRap (16).In experiments where we monitored PI(4,5)P...