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BackgroundLittle research exists regarding the relationships between green space and postpartum depression (PPD). We aimed to investigate the relationships between PPD and green space exposure, and the mediating role of physical activity (PA).MethodsClinical data were obtained from Kaiser Permanente Southern California electronic health records in 2008-2018. PPD ascertainment was based on both diagnostic codes and prescription medications. Maternal residential green space exposures were assessed using street view-based measures and vegetation types (i.e., street tree, low-lying vegetation, and grass), satellite-based measures [i.e., Normalized Difference Vegetation Index (NDVI), land-cover green space, and tree canopy cover], and proximity to the nearest park. Multilevel logistic regression was applied to estimate the association between green space and PPD. A causal mediation analysis was performed to estimate the proportion mediated by PA during pregnancy in the total effects of green space on PPD.FindingsIn total, we included 415,020 participants (30.2 ± 5.8 years) with 43,399 (10.5%) PPD cases. Hispanic mothers accounted for about half of the total population. A reduced risk for PPD was associated with total green space exposure based on street-view measure [500 m buffer, adjusted odds ratio (OR) per interquartile range: 0.98, 95% CI: 0.97-0.99], but not NDVI, land-cover greenness, or proximity to a park. Compared to other types of green space, tree coverage showed stronger protective effects (500 m buffer, OR = 0.98, 95% CI: 0.97-0.99). The proportions of mediation effects attributable to PA during pregnancy ranged from 2.7% to 7.2% across green space indicators.InterpretationStreet view-based green space and tree coverage were associated with a decreased risk of PPD. The observed association was primarily due to increased tree coverage, rather than low-lying vegetation or grass. Increased PA was a plausible pathway linking green space to lo

Few studies have assessed extreme temperatures' impact on gestational diabetes mellitus (GDM). We examined the relation between GDM risk with weekly exposure to extreme high and low temperatures during the first 24 weeks of gestation and assessed potential effect modification by microclimate indicators.MethodsWe utilized 2008-2018 data for pregnant women from Kaiser Permanente Southern California electronic health records. GDM screening occurred between 24 and 28 gestational weeks for most women using the Carpenter-Coustan criteria or the International Association of Diabetes and Pregnancy Study Groups criteria. Daily maximum, minimum, and mean temperature data were linked to participants' residential address. We utilized distributed lag models, which assessed the lag from the first to the corresponding week, with logistic regression models to examine the exposure-lag-response associations between the 12 weekly extreme temperature exposures and GDM risk. We used the relative risk due to interaction (RERI) to estimate the additive modification of microclimate indicators on the relation between extreme temperature and GDM risk.ResultsGDM risks increased with extreme low temperature during gestational weeks 20--24 and with extreme high temperature at weeks 11-16. Microclimate indicators modified the influence of extreme temperatures on GDM risk. For example, there were positive RERIs for high-temperature extremes and less greenness, and a negative RERI for low-temperature extremes and increased impervious surface percentage.DiscussionSusceptibility windows to extreme temperatures during pregnancy were observed. Modifiable microclimate indicators were identified that may attenuate temperature exposures during these windows, which could in turn reduce the health burden from GDM.

Jean de Dieu Gatete,1,2,* Charlita C Worthy,1,* Ram Jagannathan,3 Christopher W DuBose,1 David B Sacks,4 Anne E Sumner1,5,6 1Section on Ethnicity and Health, Diabetes, Endocrinology, and Obesity Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA; 2Institute of Global Health Equity, University of Global Health Equity, Kigali, Rwanda; 3Emory Global Diabetes Research Center of Woodruff Health Sciences Center and Emory University, Rollins School of Public Health of Emory University, Atlanta, GA, USA; 4Clinical Center, National Institutes of Health, Bethesda, MD, USA; 5Hypertension in Africa Research Team, North-West University, Potchefstroom, North-West, South Africa; 6National Institute of Minority Health and Health Disparities, Bethesda, MD, USA*These authors contributed equally to this workCorrespondence: Anne E Sumner, National Institutes of Health, Bld 10-CRC, Rm 6-5940, MSC 1612, Bethesda, MD, 20892-1612, USA, Tel +1-301-402-4240, Email anne.sumner@nih.govObjective: To improve detection of abnormal glucose tolerance (Abnl-GT), attention has moved beyond the oral glucose tolerance test (OGTT), to non-fasting markers of glycemia, specifically, HbA1c, fructosamine (FA) and glycated albumin (GA). Emerging data suggest that in African descent populations, the combination of HbA1c and GA is superior to the combination of HbA1c and FA. However, the diagnosis of Abnl-GT is usually based on tests which are performed only once. As reproducibility of Abnl-GT diagnosis by HbA1c, fructosamine (FA) and glycated albumin (GA) is unknown, reproducibility of Abnl-GT diagnosis by HbA1c, FA and GA were assessed in 209 African-born Blacks living in America.Methods: At Visits 1 and 2 (9 ± 4 days apart), samples were obtained for HbA1c, FA and GA levels. Glucose tolerance status was determined at Visit 1 by OGTT. Reproducibility was based on the К-statistic and paired t-tests. Threshold

RAS-ERK (extracellular signal–regulated kinase) pathway signals are modulated by scaffold proteins that assemble the components of different kinase tiers into a sequential phosphorylation cascade. In the prevailing model scaffold proteins function as isolated entities, where the flux of phosphorylation events progresses downstream linearly, to achieve ERK phosphorylation. We show that different types of scaffold proteins, specifically KSR1 (kinase suppressor of Ras 1) and IQGAP1 (IQ motif-containing guanosine triphosphatase activating protein 1), can bind to each other, forming a complex whereby phosphorylation reactions occur across both species. MEK (mitogen-activated protein kinase kinase) bound to IQGAP1 can phosphorylate ERK docked at KSR1, a process that we have named “trans-phosphorylation.” We also reveal that ERK trans-phosphorylation participates in KSR1-regulated adipogenesis, and it also underlies the modest cytotoxicity exhibited by KSR-directed inhibitors. Overall, we identify interactions between scaffold proteins and trans-phosphorylation as an additional level of regulation in the ERK cascade, with broad implications in signaling and the design of scaffold protein–aimed therapeutics.

ERK 1/2 proteregulatory proteins like scaffold proteins, that link the components of MAP kinases sequential tiers to form a multienzymatic complex. This way ERK signals are fine-tune (amplitude, intensity and duration), afford signal fidelity and have spatial selectivity. Some examples of scaffold proteins are KSR1, that bind C/BRAF, MEK1/2 and ERK1/2, and IQGAP1, that binds BRAF, MEK1/2 AND ERK1/2. In both cases depletion or blockade of this scaffold functions prevents RAS-ERK pathway-driven neoplasia. We demostrated that ERK can binds constitutively to scaffold, to be cis-phosphorylated therein by scaffold-bound MEK which would enable its binding to a free, phosphorylated ERK monomer to form a dimer. We have found that KSR1 y IQGAP1 can functionally associate with each other. As a consecuence, ERK docked at KSR1 can be phosphorylated by MEK bound to IQGAP1, a process named ¿trans-phosphorylarion¿. The results identify interactions betweeen scaffold proteins as a higher-order level of regulation in the ERK cascade. ins are major actors in the transduction of signals conveyed by external stimuli to the interior of the cell, being regulators of biochemical processes such as proliferation, differentiation or survival. ERK signals are modulated by different

The measurement of blood ketones in preference to urine ketones has become a well-established tool in the diagnosis and management of diabetic ketoacidosis (DKA). However, there remains considerable disparity between diabetes guidelines regarding if, how, and when this test should be used. While recent guidelines now mainly emphasize blood measurement, several issues nonetheless remain. Many laboratories still measure blood ketones using a semiquantitative test that does not measure the predominant ketone, beta-hydroxybutyrate (BOHB), which may hinder patient management. Even when BOHB is measured, the evidence for cutoffs used in DKA diagnosis or exclusion is limited, while its use in gauging severity, treatment progress, and resolution is not fully clear. Lastly, although employing point-of-care meters instead of a laboratory for BOHB measurement brings undoubted benefits, this approach has its own challenges. This article provides a perspective on these topics to complement current recommendations and to suggest how future research may improve its use in the DKA context., Correction:Controversies Around the Measurement of Blood Ketones to Diagnose and Manage Diabetic Ketoacidosis (vol 45, pg 267, 2022)Diabetes Care. Volume: 45, Issue: 6, Page: 1490-1490DOI: 10.2337/dc22-er06aWOS: 000886431800031Scopus: 2-s2.0-85131270352PubMed ID: 35239959

Background: Visceral leishmaniasis, also known on the Indian subcontinent as kala-azar, is a fatal form of leishmaniasis caused by the protozoan parasite Leishmania donovani and transmitted by the bites of the vector sandfly Phlebotomus argentipes. To achieve and sustain elimination of visceral leishmaniasis, the transmission potential of individuals exposed to L donovani from across the infection spectrum needs to be elucidated. The aim of this study was to evaluate the relative infectiousness to the sandfly vector of patients with visceral leishmaniasis or post-kala-azar dermal leishmaniasis, before and after treatment, and individuals with asymptomatic infection. Methods: In this prospective xenodiagnosis study done in Muzaffarpur district of Bihar, India, we included patients with clinically confirmed active visceral leishmaniasis or post-kala-azar dermal leishmaniasis who presented to the Kala-Azar Medical Research Center. These participants received treatment for L donovani infection. We also included asymptomatic individuals identified through a serosurvey of 17 254 people living in 26 high-transmission clusters. Eligible participants were aged 12–64 years, were HIV negative, and had clinically or serologically confirmed L donovani infection. During xenodiagnosis, the forearms or lower legs of participants were exposed to 30–35 female P argentipes sandflies for 30 min. Blood-engorged flies were held in an environmental cabinet at 28°C and 85% humidity for 60–72 h, after which flies were dissected and evaluated for L donovani infection by microscopy and quantitative PCR (qPCR). The primary endpoint was the proportion of participants with visceral leishmaniasis or post-kala-azar dermal leishmaniasis, before and after treatment, as well as asymptomatic individuals, who were infectious to sandflies, with a participant considered infectious if promastigotes were observed in one or more individual flies by microscopy, or if one or more of the pools of flies tested

BackgroundRecent studies have reported inconsistent associations between maternal residential green space and preterm birth (PTB, born < 37 completed gestational weeks). In addition, windows of susceptibility during pregnancy have not been explored and potential interactions of green space with air pollution exposures during pregnancy are still unclear.ObjectivesTo evaluate the relationships between green space and PTB, identify windows of susceptibility, and explore potential interactions between green space and air pollution.MethodsBirth certificate records for all births in California (2001-2008) were obtained. The Normalized Difference Vegetation Index (NDVI) was used to characterized green space exposure. Gestational age was treated as a time-to-event outcome; Cox proportional hazard models were applied to estimate the association between green space exposure and PTB, moderately PTB (MPTB, gestational age < 35 weeks), and very PTB (VPTB, gestational age < 30 weeks), after controlling for maternal age, race/ethnicity, education, and median household income. Month-specific green space exposure was used to identify potential windows of susceptibility. Potential interactions between green space and air pollution [fine particulate matter < 2.5 µm (PM2.5), nitrogen dioxide (NO2), and ozone (O3)] were examined on both additive and multiplicative scales.ResultsIn total, 3,753,799 eligible births were identified, including 341,123 (9.09%) PTBs, 124,631 (3.32%) MPTBs, and 22,313 (0.59%) VPTBs. A reduced risk of PTB was associated with increases in residential NDVI exposure in 250 m, 500 m, 1000 m, and 2000 m buffers. In the 2000 m buffer, the association was strongest for VPTB [adjusted hazard ratio (HR) per interquartile range increase in NDVI: 0.959, 95% confidence interval (CI): 0.942-0.976)], followed by MPTB (HR = 0.970, 95% CI: 0.962-0.978) and overall PTB (HR = 0.972, 95% CI: 0.

BACKGROUND: Recent studies have reported inconsistent associations between maternal residential green space and preterm birth (PTB, born < 37 completed gestational weeks). In addition, windows of susceptibility during pregnancy have not been explored and potential interactions of green space with air pollution exposures during pregnancy are still unclear. OBJECTIVES: To evaluate the relationships between green space and PTB, identify windows of susceptibility, and explore potential interactions between green space and air pollution. METHODS: Birth certificate records for all births in California (2001-2008) were obtained. The Normalized Difference Vegetation Index (NDVI) was used to characterized green space exposure. Gestational age was treated as a time-to-event outcome; Cox proportional hazard models were applied to estimate the association between green space exposure and PTB, moderately PTB (MPTB, gestational age < 35 weeks), and very PTB (VPTB, gestational age < 30 weeks), after controlling for maternal age, race/ethnicity, education, and median household income. Month-specific green space exposure was used to identify potential windows of susceptibility. Potential interactions between green space and air pollution [fine particulate matter < 2.5 µm (PM2.5), nitrogen dioxide (NO2), and ozone (O3)] were examined on both additive and multiplicative scales. RESULTS: In total, 3,753,799 eligible births were identified, including 341,123 (9.09%) PTBs, 124,631 (3.32%) MPTBs, and 22,313 (0.59%) VPTBs. A reduced risk of PTB was associated with increases in residential NDVI exposure in 250 m, 500 m, 1000 m, and 2000 m buffers. In the 2000 m buffer, the association was strongest for VPTB [adjusted hazard ratio (HR) per interquartile range increase in NDVI: 0.959, 95% confidence interval (CI): 0.942-0.976)], followed by MPTB (HR = 0.970, 95% CI: 0.962-0.978) and overall PTB (HR = 0.972

Maria Gabriela Castro,1 Melanie Dicks,2 Keisa Fallin-Bennett,1 Carol Hustedde,1 David Sacks,1 Lynn Joyce Hunter,3 William Elder1 1Department of Family and Community Medicine, University of Kentucky, Lexington, KY, USA; 2College of Pharmacy, University of Kentucky, Lexington, KY, USA; 3College of Social Work, University of Kentucky, Lexington, KY, USA Purpose: Transformation of care teaching is often didactic and conceptual instead of practical and operational. Clinical environments, slow to transform, limit student exposure to key experiences that characterize transformed care. We describe the design and implementation of TEAM Clinic (Teach students, Empower patients, Act collaboratively, Meet health goals) – an early clinical learning experience to address this gap. Methods: The TEAM Clinic curriculum was based on a review of existing curricula and best practice recommendations for the transformation of care. Three key elements were selected as the focus for a low-volume, high-service clinic: patient centeredness, interprofessional collaboration and team-based care. Learners and medically and socially complex patients were recruited for voluntary participation and completed anonymous surveys about the experience during and afterward. Results: Nine first-year medical students, two first-year social work students and one pharmacy resident were integrated into the interprofessional team. Students were assigned roles adapted to their level and skill set; deliberate interprofessional pairing was assigned to broaden perspectives on scope and role of team members. Upon completion of this two-semester experience, 11 of the 12 learners returned surveys; all rated the experience as positive (strongly agree or agree) on the Authentic Clinical Interprofessional Experience – Evaluation of Interprofessional Site tool. Patient surveys indicated satisfaction with multiple aspects of the visit. Conclusion: TEAM Clinic provided a practical example of transformati

IQ motif-containing GTPase-activating proteins (IQGAPs) are scaffolding proteins playing central roles in cell-cell adhesion, polarity, and motility. The Rho GTPases Cdc42 and Rac1, in their GTP-bound active forms, interact with all three human IQGAPs. The IQGAP-Cdc42 interaction promotes metastasis by enhancing actin polymerization. However, despite their high sequence identity, Cdc42 and Rac1 differ in their interactions with IQGAP. Two Cdc42 molecules can bind to the Ex-domain and the RasGAP site of the GTPase-activating protein (GAP)related domain (GRD) of IQGAP and promote IQGAP dimerization. Only one Rac1 molecule might bind to the RasGAP site of GRD and may not facilitate the dimerization, and the exact mechanism of Cdc42 and Rac1 binding to IQGAP is unclear. Using all-atom molecular dynamics simulations, site-directed mutagenesis, and Western blotting, we unraveled the detailed mechanisms of Cdc42 and Rac1 interactions with IQGAP2. We observed that Cdc42 binding to the Ex-domain of GRD of IQGAP2 (GRD2) releases the Ex-domain at the C-terminal region of GRD2, facilitating IQGAP2 dimerization. Cdc42 binding to the Ex-domain promoted allosteric changes in the RasGAP site, providing a binding site for the second Cdc42 in the RasGAP site. Of note, the Cdc42 "insert loop" was important for the interaction of the first Cdc42 with the Ex-domain. By contrast, differences in Rac1 insert-loop sequence and structure precluded its interaction with the Ex-domain. Rac1 could bind only to the RasGAP site of apo-GRD2 and could not facilitate IQGAP2 dimerization. Our detailed mechanistic insights help decipher how Cdc42 can stimulate actin polymerization in metastasis., Federal funds from the Frederick National Laboratory for Cancer Research, National Institutes of Health; Intramural Research Program of National Institutes of Health; Frederick National Laboratory; Center for Cancer Research; Intramural Research Program of the National Institutes of Health Clinical Center; Scientific and Technological Research Council of Turkey (TÜBİTAK)

Phosphatidylinositol 4,5 bisphosphate (PIP 2) is a key lipid messenger for regulation of cell migration. PIP 2 modulates many effectors, but the specificity of PIP 2 signalling can be defined by interactions of PIP 2-generating enzymes with PIP 2 effectors. Here, we show that type Iγ phosphatidylinositol 4-phosphate 5-kinase (PIPKIγ) interacts with the cytoskeleton regulator, IQGAP1, and modulates IQGAP1 function in migration. We reveal that PIPKIγ is required for IQGAP1 recruitment to the leading edge membrane in response to integrin or growth factor receptor activation. Moreover, IQGAP1 is a PIP 2 effector that directly binds PIP 2 through a polybasic motif and PIP 2 binding activates IQGAP1, facilitating actin polymerization. IQGAP1 mutants that lack PIPKIγ or PIP 2 binding lose the ability to control directional cell migration. Collectively, these data reveal a synergy between PIPKIγ and IQGAP1 in the control of cell migration. © 2013 European Molecular Biology Organization.

Wnt signaling pathways regulate proliferation, motility, and survival in a variety of human cell types. Dickkopf-1 (Dkk-1) is a secreted Wnt antagonist that has been proposed to regulate tissue homeostasis in the intestine. In this report, we show that Dkk-1 is secreted by intestinal epithelial cells after wounding and that it inhibits cell migration by attenuating the directional orientation of migrating epithelial cells. Dkk-1 exposure induced mislocalized activation of Cdc42 in migrating cells, which coincided with a displacement of the polarity protein Par6 from the leading edge. Consequently, the relocation of the microtubule organizing center and the Golgi apparatus in the direction of migration was significantly and persistently inhibited in the presence of Dkk-1. Small interfering RNA-induced down-regulation of Dkk-1 confirmed that extracellular exposure to Dkk-1 was required for this effect. Together, these data demonstrate a novel role of Dkk-1 in the regulation of directional polarization of migrating intestinal epithelial cells, which contributes to the effect of Dkk-1 on wound closure in vivo., Koch, Stefan Capaldo, Christopher T Samarin, Stanislav Nava, Porfirio Neumaier, Irmgard Skerra, Arne Sacks, David B Parkos, Charles A Nusrat, Asma eng R01 DK079392/DK/NIDDK NIH HHS/ R29 DK055679/DK/NIDDK NIH HHS/ DK-72564/DK/NIDDK NIH HHS/ DK-055679/DK/NIDDK NIH HHS/ R01 DK072564/DK/NIDDK NIH HHS/ DK-61379/DK/NIDDK NIH HHS/ DK-79392/DK/NIDDK NIH HHS/ R01 DK055679/DK/NIDDK NIH HHS/ R01 DK061379/DK/NIDDK NIH HHS/ Research Support, N.I.H., Extramural 2009/09/25 06:00 Mol Biol Cell. 2009 Nov;20(22):4816-25. doi: 10.1091/mbc.E09-05-0415. Epub 2009 Sep 23.

The purpose of this proposal is to evaluate the role of IQGAP1 in neoplastic transformation and metastasis of breast epithelial cells. The main emphasis is on whether IQGAP1 Is involved in invasion and metastasis of transformed breast epithelial cells, as well as the possible involvement of 1QGAP1 in regulating beta-catenin function. Major findings to date are: (i)there is a high level of expression of IQGAP1 in breast epithelium; (ii)overexpression of IQGAP1 in mammalian cells enhances cell migration and proliferation; and (iii) decreased 1QGAP1 protein levels reduced cell motility. These data reveal that IQGAP1 has a fundamental role in cell motility and invasion in breast epithelium. This information could have potential therapeutic implications in patients with breast cancer.

The purpose of this proposal is to evaluate the role of IQGAPl in neoplastic transformation and metastasis of breast epithelial cells. The main emphasis is on whether IQGAP is involved in invasion and metastasis of transformed breast epithelial cells, as well as the possible involvement of IQGAPl in regulating b-catenin function. Major findings to date are: (i) there is a high level of expression of IQGAPl in breast epithelium; and (ii) overexpression of IQGAP1 in mammalian cells enhances cell migration and proliferation. These data reveal that IQGAPl has a fundamental role in cell motility and invasion in breast epithelium. This information could have potential therapeutic implications in patients with breast cancer.

The purpose of this proposal is to evaluate the participation of Ca2+ and calmodulin in estrogen receptor (ER) function and tamoxifen resistance. The focus is directed towards the interaction between calmodulin and ER in ER signaling, as well as the possible involvement of Ca2+ and calmodulin in tamoxifen resistance. Major findings to date are: (i) calmodulin binds directly to ER and stabilizes the receptor; (ii) calmodulin protects ER from degradation in the proteasome pathway; (iii) calmodulin is necessary for estrogen-stimulated transcriptional activation by ER; (iv) the effect of calmodulin in ER transcriptional activation is independent of its effect on ER stability; and (v) inhibition of calmodulin function in the nucleus, but not at the plasma membrane, eliminates estrogen-induced transcriptional activation by ER. These data reveal that calmodulin is a component of both ER stability and ER transcriptional activity. This information could have potential therapeutic implications in patients with breast cancer., The original document contains color images. All DTIC reproductions will be in black and white.

The endothelial nitric-oxide synthase (eNOS) is a key determinant of vascular homeostasis. Like all known nitric-oxide synthases, eNOS enzyme activity is dependent on Ca2+-calmodulin. eNOS is dynamically targeted to specialized cell surface signal-transducing domains termed plasmalemmal caveolae and interacts with caveolin, an integral membrane protein that comprises a key structural component of caveolae. We have previously reported that the association between eNOS and caveolin is quantitative and tissue-specific (Feron, O., Belhassen, L., Kobzick, L., Smith, T. W., Kelly, R. A., and Michel, T. (1996) J. Biol. Chem. 271, 22810-22814). We now report that in endothelial cells the interaction between eNOS and caveolin is importantly regulated by Ca2+-calmodulin. Addition of calmodulin disrupts the heteromeric complex formed between eNOS and caveolin in a Ca2+-dependent fashion. In addition, overexpression of caveolin markedly attenuates eNOS enzyme activity, but this inhibition is reversed by purified calmodulin. Caveolin overexpression does not affect the activity of the other NOS isoforms, suggesting eNOS-specific inhibition of NO synthase by caveolin. We propose a model of reciprocal regulation of eNOS in endothelial cells wherein the inhibitory eNOS-caveolin complex is disrupted by binding of Ca2+-calmodulin to eNOS, leading to enzyme activation. These findings may have broad implications for the regulation of Ca2+-dependent signal transduction in plasmalemmal caveolae.

Plasma-membrane-associated protein kinase(s) from normal rat liver phosphorylates exogenous bovine brain calmodulin in the absence of Ca2+ and in the presence of histone or poly(L-lysine). Maximum levels of calmodulin phosphorylation are obtained at a poly(L-lysine)/calmodulin molar ratio of 0.4. Phosphoamino acid analysis revealed that calmodulin is phosphorylated on serine, threonine and tyrosine residues. Endogenous plasma-membrane-associated calmodulin was also phosphorylated by plasma-membrane-associated protein kinase(s) in the absence of added cationic protein or polypeptide. The identity of endogenous phosphocalmodulin was confirmed by immunoprecipitation with a specific anti-calmodulin monoclonal antibody. Ehrlich ascites tumor cell plasma membranes do not contain endogenous calmodulin. However, membrane-associated protein kinase(s) from these tumor cells phosphorylates bovine brain calmodulin in the presence of poly(L-lysine). These data demonstrate that phosphocalmodulin is present in liver plasma membranes and suggest that this post-translational modification could have a physiological role in this location.

An epidermal-growth-factor(EGF)-receptor preparation isolated by calmodulin-affinity chromatography from rat liver plasma membranes is able to phosphorylate calmodulin. Calmodulin phosphorylation was enhanced 3–8-fold by EGF, was dependent on the presence of a polycation or basic protein and was inhibited by micromolar concentrations of Ca2+. Phosphate incorporation into calmodulin occurs predominantly on tyrosine residues. Partial proteolysis of phosphocalmodulin by thrombin identifies Tyr99, located in the third calcium-binding domain of calmodulin, as the phosphorylated residue. Stoichiometric measurements show a 32P/calmodulin molar ratio of approximately 1 when optimal phosphorylation conditions are used.