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...

Stromal interaction molecule 1 (STIM1) has recently been identified by our group and others as an endoplasmic reticulum (ER) Ca 2؉ sensor that responds to ER Ca 2؉ store depletion and activates Ca 2؉ channels in the plasma membrane (PM). The molecular mechanism by which STIM1 transduces signals from the ER lumen to the PM is not yet understood. Here we developed a live-cell FRET approach and show that STIM1 forms oligomers within 5 s after Ca 2؉ store depletion. These oligomers rapidly dissociated when ER Ca 2؉ stores were refilled. We further show that STIM1 formed oligomers before its translocation within the ER network to ER-PM junctions. A mutant STIM1 lacking the C-terminal polybasic PM-targeting motif oligomerized after Ca 2؉ store depletion but failed to form puncta at ER-PM junctions. Using fluorescence recovery after photobleaching measurements to monitor STIM1 mobility, we show that STIM1 oligomers translocate on average only 2 m to reach ER-PM junctions, arguing that STIM1 ER-to-PM signaling is a local process that is suitable for generating cytosolic Ca 2؉ gradients. Together, our live-cell measurements dissect the STIM1 ERto-PM signaling relay into four sequential steps: (i) dissociation of Ca 2؉ , (ii) rapid oligomerization, (iii) spatially restricted translocation to nearby ER-PM junctions, and (iv) activation of PM Ca 2؉ channels.Ca 2ϩ release-activated Ca 2ϩ ͉ fluorescence recovery after photobleaching ͉ FRET ͉ store-operated Ca 2ϩ influx C a 2ϩ signals are used by cells for transducing receptor or electrical inputs into functional outputs such as gene expression, contraction, and secretion (1). Although endoplasmic reticulum (ER) Ca 2ϩ stores are the main source for inositol trisphosphate-induced transient Ca 2ϩ signals, a necessary step for activation of T lymphocytes, mast cells, and many other cells is a persistent increase in Ca 2ϩ concentration (2). These sustained Ca 2ϩ signals require retrograde signaling from the lumen of the ER to Ca 2ϩ channels in the plasma membrane (PM) in a process called store-operated Ca 2ϩ (SOC) influx [also termed Ca 2ϩ release-activated Ca 2ϩ (CRAC) in immune cells] (3). The Stauderman/Cahalan groups and our group have recently identified the single transmembrane protein stromal interaction molecule 1 (STIM1) as an ER Ca 2ϩ sensor that responds to the depletion of ER Ca 2ϩ and activates SOC/CRAC channels in the PM (4-6). STIM1 senses Ca 2ϩ by an EF hand Ca 2ϩ -binding site in the lumen of the ER (7). A recombinant fragment of the luminal region of STIM1 has been shown to form dimers and oligomers in the absence of Ca 2ϩ in vitro (8). Overexpression of STIM1 mutants with a disrupted EF hand Ca 2ϩ -binding motif resulted in constitutive activation of Ca 2ϩ influx (5, 6, 9). It was also observed that depletion of ER Ca 2ϩ stores induced STIM1 translocation to punctate structures near the PM that correspond to ER-PM junctions (5, 10). STIM1 colocalizes at these junctions with the CRAC channel Orai1 (also known as CRACM1) and synergistically activates SOC/CRAC infl...

Although phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is a well-characterized precursor for the second messengers inositol 1,4,5-trisphosphate, diacylglycerol [1] and phosphatidylinositol 3,4,5-trisphosphate [2], it also interacts with the actin-binding proteins profilin and gelsolin [3], as well as with many signaling molecules that contain pleckstrin homology (PH) domains [4]. It is conceivable that stimuli received by receptors in the plasma membrane could be sufficiently strong to decrease the PtdIns(4,5)P2 concentration; this decrease could alter the structure of the cortical cytoskeleton and modulate the activity of signaling molecules that have PH domains. Here, we tested this hypothesis by using an in vivo fluorescent indicator for PtdIns(4,5)P2, by tagging the PH domain of phospholipase C delta 1 (PLC-delta 1) with the green fluorescent protein (GFP-PH). When expressed in cells, GFP-PH was found to be enriched at the plasma membrane. Binding studies in vitro and mutant analysis suggested that GFP-PH bound PtdIns(4,5)P2 selectively over other phosphatidylinositol lipids. Strikingly, receptor stimulation induced a transient dissociation of GFP-PH from the plasma membrane, suggesting that the concentration of PtdIns(4,5)P2 in the plasma membrane was effectively lowered. This transient dissociation was blocked by the PLC inhibitor U73122 but was not affected by the phosphoinositide (PI) 3-kinase inhibitor wortmannin, suggesting that it is mostly mediated by PLC and not by PI 3-kinase activation. Overall, our studies show that PtdIns(4,5)P2 can have second messenger functions of its own, by mediating a transient dissociation of proteins anchored in the plasma membrane.

Calcium-calmodulin-dependent protein kinase II (CaMKII) is thought to increase synaptic strength by phosphorylating postsynaptic density (PSD) ion channels and signaling proteins. It is shown that N-methyl-D-aspartate (NMDA) receptor stimulation reversibly translocates green fluorescent protein-tagged CaMKII from an F-actin-bound to a PSD-bound state. The translocation time was controlled by the ratio of expressed beta-CaMKII to alpha-CaMKII isoforms. Although F-actin dissociation into the cytosol required autophosphorylation of or calcium-calmodulin binding to beta-CaMKII, PSD translocation required binding of calcium-calmodulin to either the alpha- or beta-CaMKII subunits. Autophosphorylation of CaMKII indirectly prolongs its PSD localization by increasing the calmodulin-binding affinity.