Release from tonic inhibition of T cell activation through transient displacement of C-terminal Src kinase (Csk) from lipid rafts.

In resting peripheral T cells, Csk is constitutively present in lipid rafts through an interaction with the Csk SH2-binding protein, PAG, also known as Cbp. Upon triggering of the T cell antigen receptor (TCR), PAG/Cbp is rapidly dephosphorylated leading to dissociation of Csk from lipid rafts. However, tyrosine phosphorylation of PAG/Cbp resumes after 3--5 min, at which time Csk reassociates with the rafts. Cells overexpressing a mutant Csk that lacks the catalytic domain, but displaces endogenous Csk from lipid rafts, have elevated basal levels of TCR-zeta-chain phosphorylation and spontaneous activation of an NFAT-AP1 reporter from the proximal interleukin-2 promoter as well as stronger and more sustained responses to TCR triggering than controls. We suggest that a transient release from Csk-mediated inhibition by displacement of Csk from lipid rafts is important for normal T cell activation.

Activation of the COOH-terminal Src kinase (Csk) by cAMP-dependent protein kinase inhibits signaling through the T cell receptor.

In T cells, cAMP-dependent protein kinase (PKA) type I colocalizes with the T cell receptor-CD3 complex (TCR/CD3) and inhibits T cell function via a previously unknown proximal target. Here we examine the mechanism for this PKA-mediated immunomodulation. cAMP treatment of Jurkat and normal T cells reduces Lck-mediated tyrosine phosphorylation of the TCR/CD3 zeta chain after T cell activation, and decreases Lck activity. Phosphorylation of residue Y505 in Lck by COOH-terminal Src kinase (Csk), which negatively regulates Lck, is essential for the inhibitory effect of cAMP on zeta chain phosphorylation. PKA phosphorylates Csk at S364 in vitro and in vivo leading to a two- to fourfold increase in Csk activity that is necessary for cAMP-mediated inhibition of TCR-induced interleukin 2 secretion. Both PKA type I and Csk are targeted to lipid rafts where proximal T cell activation occurs, and phosphorylation of raft-associated Lck by Csk is increased in cells treated with forskolin. We propose a mechanism whereby PKA through activation of Csk intersects signaling by Src kinases and inhibits T cell activation.

PKAI as a potential target for therapeutic intervention.

We have mapped a molecular mechanism for the impaired T-cell function in HIV infection and common variable immunodeficiency (CVI). Protein kinase A type I (PKAI) has a key role as an inhibitor of immune function in T lymphocytes and is activated following antigen receptor triggering. T cells from patients with HIV infection and CVI have increased activation of PKAI. This inhibits immune function and proliferation of T cells. Selective antagonists that block cAMP action through PKAI improve the immune function of T cells from HIV-infected patients up to 300%. Furthermore, combination of cAMP antagonists with interleukin-2 normalized immune responses of T cells from all patients examined and stimulated immune function of T cells from HIV-infected patients up to 600%. In addition, in vitro experiments indicate that approximately 50% of patients with CVI have a T-cell dysfunction that might benefit from a treatment reversing PKAI hyperactivation. This outlines PKAI as a potentially attractive drug target for immunomodulating therapy in HIV infection, as well as for the treatment of other immunodeficiency disorders such as CVI.

Kinetic properties of the C-terminal Src kinase, p50csk.

Csk is an important regulator of tyrosine kinases of the Src family. In this paper, we have characterised the kinetics and catalytic properties of a highly active and stable enzyme obtained in milligram amounts by expressing the enzyme as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli. Using the synthetic polyamino acid poly(Glu, Tyr) as substrate, phosphotransferase activity was linear for 7-8 min with Mg2+ and 5 min with Mn2+. With Mg2+ and Mn2+, respectively, K(m) (ATP) was 56.9 +/- 6.2 and 5.4 +/- 0.6 microM and Vmax was 293 +/- 52 and 217 +/- 38 pmol phosphate transferred (microgram Csk)-1 min-1. Optimal concentrations of Mg2+ and Mn2+ were 4-10 mM and 2-3 mM, respectively, and higher concentrations of both cations were inhibitory. The Csk activity was highly sensitive to monovalent (Na+, K+) and divalent (Ca2+) cations, the sensitivity being 2-5-fold higher with Mg2+ than Mn2+. Physiological concentrations of Ca2+ (less than 10 microM) were without effect. Autophosphorylation of Csk was demonstrated in vitro, but did not influence the catalytic activity. Addition of inorganic phosphate above 100 microM strongly inhibited Csk catalytic activity towards poly(Glu, Tyr) in the presence of Mn2+, but not in the presence of Mg2+. Phosphorylation of a physiological substrate (Lck) and autophosphorylation of Csk was not inhibited by phosphate, indicating that the phosphate-dependent inhibition of Csk activity was substrate specific.

Structure, function, and regulation of human cAMP-dependent protein kinases.

A large number of hormones, neurotransmitters, and other signaling substances that bind to G-protein-coupled cell-surface receptors have their signals converge at one sole second messenger, cAMP. The question of how specificity can be maintained in a signal-transduction system in which many extracellular signals leading to a vast array of intracellular responses are all mediated through one second-messenger system has been the subject of thorough investigation and a great deal of speculation. An increasing number of cAK isozymes, consisting of homo- or heterodimers of R subunits (RIalpha, RIbeta, RIIalpha, RIIbeta) with associated catalytic subunits (C alpha, Cbeta, Cgamma), may, at least in part, explain this specificity. The various cAK isozymes display distinct biochemical properties, and the heterogeneous subunits of cAK reveal cell-specific expression and differential regulation at the level of gene transcription, mRNA stability, and protein stability in response to a wide range of hormones and other signaling substances. The existence of a number of anchoring proteins specific to either RIIalpha or RIIbeta, and which localize cAKII isozymes toward distinct substrates at defined subcellular loci, strongly supports the idea that specific functions can be assigned to the various cAK isozymes. The demonstration that selective activation of cAKI is necessary and sufficient for cAMP-mediated inhibition of T-cell proliferation, and the observation that T-cell activation is associated with redistribution and colocalization of cAKI to the TCR, is also compatible with the notion of isozyme-specific effects.

Medication compliance, antidepressant blood levels, and side effects in Southeast Asian patients.

A study of medication compliance, side effects, and clinical change with the use of antidepressants in 32 Southeast Asian refugee patients seen at an urban mental health center is reported. Patients met criteria for either major depressive episode, posttraumatic stress disorder, or both. Only five of the 32 patients who stated that they were taking their medications regularly had antidepressant blood levels in the therapeutic range. Another 10 patients had subtherapeutic levels and the remaining 17 had undetectable blood levels. Patients with therapeutic blood levels had fewer side effects (p = 0.049) than patients with undetectable blood levels. Blood levels tended (p = 0.070) to be correlated with clinical improvement. The authors discuss cultural attitudes of Southeast Asian refugee patients toward medication use and side effects that appear to influence medication compliance.

Depression and posttraumatic stress disorder in Southeast Asian refugees.

The authors report on 404 Southeast Asian refugees seen at a community clinic. Approximately three-quarters of these patients met DSM-III criteria for major depressive episode, and 14% had posttraumatic stress disorder. Complaints of pain and sleep disturbances were the predominant presenting symptoms. Most of the men were married, but more than 40% of the women were widowed. Between 15% and 30% of the patients reported specific traumatic experiences either in their homeland or during their escape. Widowhood and such traumatic experiences were positively correlated with more symptoms of depression and anxiety.

Acculturation and mental health: a study of Hmong refugees at 1.5 and 3.5 years postmigration.

This study of Hmong refugees at 1.5 and 3.5 years following arrival in the United States showed considerable improvement on psychiatric self-rating scales. Social changes over the 2 year interim (including a high unemployment rate) were few. Earlier premigration and postmigration variables correlated with high symptom levels at 1.5 years were not correlated with these symptoms at 3.5 years. Events in the acculturation process which accompany, and perhaps account for some of these observations are indicated.

Symptom change over time among Hmong refugees: psychiatric patients versus nonpatients.

Most Hmong refugees in Minnesota were assessed in September, 1977, using two self-rating scales (Zung Scale for Depression and 90-item Symptom Checklist). The same group was restudied two years later. During 12 months of 1977-78, psychiatric services were offered to this population. Those seeking psychiatric care were compared with those who did not. These data indicate that the patients reported considerably more symptoms than nonpatients in 1977 prior to receiving care. The patients showed more improvement than the nonpatients between 1977 and 1979.

Refugees who do and do not seek psychiatric care. An analysis of premigratory and postmigratory characteristics.

Social psychiatric research can provide information about the role of interpersonal and societal factors in the genesis of psychiatric disorder. This discipline relies heavily on "experiments in nature" which expose a large number of people to a potentially pathological social stimulus. It also depends in large part upon the study of nonpatients to serve as a comparative group for patients. Both conditions are met in this study of Hmong refugees from Indochina. While the population and the event are esoteric to some extent, their experiences of sudden sociocultural change, geographic migration, role discontinuity, identity crisis, and massive loss are common experiences among many psychiatric patients, regardless of their origin. Thus this study contributes to our understanding regarding the social genesis of psychiatric disorder. This prospective study of refugees to the United States was undertaken among the Hmong population in Minnesota (N = 97) during 1977. Subsequently 17 of this group became psychiatric patients over a 12-month period. Premigration and postmigration factors associated with patient status are described. Hypotheses are offered regarding those postmigration experiences or social strategies which favored or prevented psychiatric status.

Migration and mental health among Hmong refugees. Association of pre- and postmigration factors with self-rating scales.

To date there have been no epidemiological studies of a refugee population using self-rating scales. This method was used in a study of Hmong refugees in Minnesota. Self-reported symptoms were compared with premigration and postmigration factors to assess those characteristics associated with increased symptom reporting. Relatively few premigration factors influenced these self-reports, whereas several postmigration factors were significantly correlated with symptoms. These findings suggest certain interventions which might enhance the adjustment of subsequent refugees.